Previous studies showed that doxorubicin could lead to osteoarthritis (OA) by inducing chondrocyte inflammation and apoptosis. Besides, it is reported that platelet-rich plasma (PRP) could suppress the activation of inflammatory NF-κB signaling. Here, we aimed to determine whether PRP was able to exert a protective effect against doxorubicin-induced chondrocyte damages.To determine whether PRP protects chondrocytes against destabilization of the medial meniscus (DMM)-induced osteoarthritis, mice were treated with PRP and doxorubicin, and the cartilage destruction was observed through Safranin O-fast green staining and osteoarthritis scoring. ELISA assay was used to check the release of TNF-α and ILs. In vitro, we treated chondrocytes with doxorubicin and PRP; CCK-8 was used to measure cell viability. Western blot, real-time PCR, and ELISA were applied to check apoptosis-related signaling and inflammation-associated factors.The results from the mouse model suggested that PRP attenuated doxorubicin-induced cartilage destruction in vivo. Doxorubicin promoted chondrocyte apoptosis while PRP ameliorated this damage. PRP inhibited doxorubicin-induced dysregulation of cell matrix-related factors, including SOX9, Col2A1, Col10A1, and Aggrecan, reduced protein levels of doxorubicin-induced inflammatory markers, COX-2, and iNOS, and blocked doxorubicin-induced phosphorylation of IκB and NF-κB in articular chondrocytes.PRP improved doxorubicin-induced damage on chondrocytes. This research might provide a new theoretical basis for the clinical treatment of osteoarthritis caused by doxorubicin.The online version contains supplementary material available at 10.1186/s10020-021-00314-2. Osteoarthritis (OA) is a prevalent joint disorder globally, occurring in about 20% of people aged over 60-year old, and severely affecting the daily life of those patients . 12-week-old male C57BL/6 mice were housed in a designated mouse facility operating at a controlled temperature (24 °C) and a circadian rhythm of 12 h and had free access to clean water and foods. These mice were randomly divided into the indicated groups in corresponding experiments. OA was induced in mice by destabilizing the medial meniscus (DMM) of the knees. One week after surgery, the mice received doxorubicin (10 mg/kg) injection and/or intra-articular injection of 15 μL PRP every week. After 4 weeks, the mice were euthanized, and the knee joints and plasma were collected , TGF-β1 (DB100B), TNF-α (MTA00B), IL-1β (MLB00C) were purchased from R&D systems .The knee joint samples were collected and fixed in 4% paraformaldehyde for 24 h. The bones were placed in 10% EDTA for 1 month to perform decalcification, embedded with paraffin, prepared as 5 μm thick sections, and stained with Safranin O-fast green staining kit  following manufacturer’s instruction. The images were captured and calculated. The scores of the femur and tibia were summed and presented as the OARSI score for each sample.Mice were sacrificed, and the total articular cartilage was isolated from the femur head, cut into pieces, and digested with 1.5 mg/mL pronase for 2 h and collagenase II under shaking at 37 °C for 8 h. Cells were then filtered through a 70 μm cell strainer and washed 3 times with PBS. Subsequently, the obtained chondrocytes were placed in culture plates and maintained in DMEM/F12 media  containing 10% fetal bovine serum , 100 U/mL penicillin, and 100 μg/mL streptomycin. The media were changed every 2 days. After preparation of chondrocytes from joint articulation, toluidine blue staining and collagen type II immunohistochemical staining were carried out to identify the purity of these chondrocytes  were extracted from the bone marrow of the femur and tibia of mice and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS with M-CSF (10 ng/mL). BMMs were differentiated in a humidified chamber at 37 °C with 5% CO2.ATDC5 cells were cultured in Alpha modification of Eagle’s media containing 10% FBS, 100 U/mL penicillin, and Insulin-Transferrin-Selenium solution. All cells were cultured at 37 °C in a humidified incubator with 5% CO3 cells per well overnight and treated with doxorubicin or PRP at the indicated concentration for 24 h. At the treatment, 10 μL of CCK-8  was added to each well, and cells were continuously incubated for 1 more hour at the incubator. Then the absorbance values at 450 nm was detected by an absorbance reader.The proliferation ability of cardiomyocytes was analyzed by CCK-8 kit. In brief, cells were digested and plated into 96-well plates with a density of 1 × 10Cells were lysed in 1× ice-cold RIPA lysis buffer  and centrifuged to discard the deposition. The protein concentration was determined using BCA kits . A total of 35 μg proteins were separated on 10% SDS-polyacrylamide gel (PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 h at room temperature with 5% nonfat milk dissolved in 1× TBST and incubated with specific primary antibodies at 4 °C overnight. Then the membranes were further incubated for 2 h at room temperature with the corresponding secondary antibodies. After that, labeled proteins were visualized by an ECL kit  and captured using Gel imaging system . Primary antibodies against p-NF-κB (3033), NF-κB (3032), p-IκBα, (8219), IκBα (9242) were purchased from Cell Signaling Technology  and against β-actin (sc-1616), COX-2 (sc-376861), Sox9 (SC-166505), iNOS (SC-7271), PARP (SC-74470) and cleaved PARP (SC-56196) were purchased from SantaCruz Biotechnology  and diluted following the manufacturers’ protocols.−ΔΔCt method. The primers used in this study were MMP1 forward 5ʹ-ATACGTTGGTGGTGTTGTAATGT-3ʹ and reverse 5ʹ-GTCA CCTCTTTGGATGCCATAAA-3ʹ, MMP3 forward, 5ʹ-TTAAAGACAGGCACTTTTG GCG-3ʹ and reverse 5ʹ-CCCTCGTATAGCCCAGAACT-3ʹ, MMP13 forward 5ʹ-CTT TGGCTTAGAGGTGACTGG-3ʹ and reverse 5ʹ-AGGCACTCCACATCTTGGTTT-3ʹ, TNF-α forward 5ʹ-TGGAACACGTCGTGGGATAATG-3ʹ and reverse 5ʹ-GGCAGAC TTTGGATGCTTCTT-3ʹ, IL-6 forward 5ʹ-GGCGGATCGGATGTTGTGAT-3ʹ and reverse 5ʹ-GGACCCCAGACAATCGGTTG-3ʹ, Col2A1 forward 5ʹ-CCTCAAGGCA AAGTTGGTCCT-3ʹ and reverse 5ʹ-CTCCCGTCTCACCGTCTTTT-3ʹ, Col10A1 forward 5ʹ-GGTGTGAATGGGCGGAAAG-3ʹ and reverse 5ʹ-GCTTCCCAATACCTT CTCGTC-3ʹ, as well as Aggrecan forward 5ʹ-CCCAGGATAAAACCAGGCAG-3ʹ and reverse 5ʹ-CGGCCAAGGGTTGTAAATGG-3ʹ.Total RNAs were extracted using TRIzol Reagent  following the manufacturer’s protocols after indicated treatments. Their concentration was determined spectrophotometrically at 260 nm (Thermo-Scientific NanoDrop 2000), and their quality and purity were verified by A260/A280 ratio (1.8–2.2). Then RNAs were reverse transcribed to cDNA using a cDNA Synthesis Kit . Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Green SuperMix kit  to detect gene expression following the manufacturer’s protocols. Small endogenous nucleolar U6 snRNA and GAPDH were used as the internal controls for normalizing miR-125a-5p and MTFP1, respectively. Gene expression level was determined using the 2The data were presented as the mean ± SD. Student t test and One-way ANOVA method followed by Bonferroni post hoc test were used to assess the difference between two groups and multiple groups, separately. A p < 0.05 was considered statistically significant.To investigate whether PRP is able to exert a protective effect against doxorubicin-induced inflammation of chondrocytes, we treated mice with PRP and doxorubicin. As shown in Fig. As the extract from total blood, PRP is considered a cocktail of growth factors, among which TGF-β1 is the most abundant, presentative ingredient. In this study, we determined PRP concentration by evaluating TGF-β1 level and investigated the proliferation of ATDC5 cells and primary articular chondrocytes. The results showed that 2 ng/mL PRP treatment enhanced proliferation of ATDC5 cells  collected from blood exhibited favorable effects against doxorubicin-caused damages to articular cartilage in vivo. PRP suppressed doxorubicin-induced chondrocyte apoptosis and matrix degradation by attenuating NF-κB signaling and expression of downstream inflammatory factors iNOS, TNF-α, and ILs Fig. . Our finAdditional file 1: Figure S1. Toluidine blue staining and collagen type II immunohistochemical staining of the mouse chondrocytes.Additional file 2: Figure S2. TGF-β1 is the crucial factor in PRP responsible for its preventive effects on arthritis. Shown are the Western blot results of PARP, cleaved PARP, β-actin, p-NF-κB, and NF-κB in ATDC5 (A), primary articular chondrocytes cells (C), SW1353 (E) and BMMs (H) treated with PRP, doxorubicin, and TGF-β1 for 24 h, % of PRP action is the effect of TGF-β1 and other in ATDC5 (B), primary articular chondrocytes cells (D), SW1353 (F) and BMMs (H).


Further stability to the three-ion aggregate is provided by intramolecular amine-N—H⋯O(carboxylate) hydrogen bonds. Hydrogen bonding is also prominent in the molecular packing with amide-N—H⋯O(amide) interactions, leading to eight-membered {⋯HNCO}2 synthons, amide-N—H⋯O(water), water-O—H⋯O(carboxylate) and water-O—H⋯O(water) hydrogen bonds featuring within the three-dimensional architecture. The calculated Hirshfeld surfaces for the individual components of the asymmetric unit differentiate the water molecules owing to their distinctive supramolecular association. For each of the anion and cation, H⋯H contacts predominate  followed by H⋯O/O⋯H contacts .The title Cu Cu—N2 = 2.0016 (12) Å, being approximately 0.1 Å shorter than the Cu—N1 bond of 2.1086 (11) Å. Whereas the conformation of the five-membered chelate ring is best described as being an envelope with the C4 atom being the flap atom, the six-membered chelate ring approximates a chair conformation. The acetate anions are weakly associated with the complex cation, forming relatively long Cu⋯O3 separations of 3.2048 (15) Å with extra stability to the three-ion aggregate provided by intramolecular amine-N—H⋯(carboxylate) hydrogen bonds, Table 14O2 tetragonally distorted. From symmetry, the N1-bound carbamoylethyl groups lie to opposite sides of the CuN4 plane and the N1—C9—C10—C11 torsion angle of −178.52 (12)° is consistent with an –anti-periplanar (–ap) configuration.The molecular structure diagram showing the complex dication and loosely associated anions is shown in Fig. 1via an eight-membered {⋯HNCO}2 synthon, and the second H atom participates in an amide-N—H⋯O(water) hydrogen bond. This water molecule, i.e. water-O1W, also forms donor interactions to a carboxylate-O2 atom and to the second water molecule, i.e. water-O2W. The latter forms donor interactions with the amide-O and carboxylate-O3 atoms. As can be seen from the unit-cell diagram of Fig. 2a axis direction with the prominent hydrogen bonds between the molecules in that direction being water-O—H⋯O(water) and water-O—H⋯O3(carboxylate). The remaining hydrogen bonds extend laterally to consolidate the three-dimensional supramolecular network.Conventional hydrogen bonding is prominent among the directional supramolecular contacts evident in the crystal of (I)dnorm  and 1.131 a.u. (red). Key interatomic parameters are listed in Table 2The Hirshfeld surface analysis for each constituent of (I)dnorm-Hirshfeld surface of the O1W-water molecule are due to the formation of conventional water-O—H⋯O(water) and water-O—H⋯O(carboxylate) hydrogen bonds, Fig. 3a). The other bright-red spot appearing on the dnorm-Hirshfeld surface is due to the formation of a conventional primary amide-N—H⋯O(water) hydrogen bond, Fig. 3b). Further, weak methylene/methyl-C—H⋯O(water) interactions are also shown as faint red spots near atoms H5A, H7B and H8C in Fig. 3b). Similar to the O1W-water molecule, the two O2W-H atoms participate in conventional water-O—H⋯O(carboxylate) and water-O—H⋯O(amide) hydrogen bonds. These hydrogen bonds are manifested as two bright-red spots on the dnorm-Hirshfeld surface of the O2W molecule, Fig. 4a). The third bright red spot, evident in Fig. 4b), is due to the water-O—H⋯O(water) hydrogen bond as discussed above.As a hydrogen-bond donor, the two bright red spots on the dnorm-Hirshfeld surface correspond to the water-O—H⋯O(carboxylate) hydrogen bonds, Fig. 5a); the amide-N—H⋯O(carboxylate) hydrogen bond, which also leads to a bright-red spot, is highlighted in Fig. 5b). At the same time, the weak methylene-H4A/methyl-H7A⋯O3(carboxylate) interactions, with separations of 0.38 and 0.47 Å shorter than the sum of van der Waals radii, respectively, are shown as faint red spots in Fig. 5b).For the carboxylate anion, the bright-red spots on its dnorm-Hirshfeld surface calculated for the cation, the bright-red spots near the amide-O1, methyl-H7A, amine-H1N and amide-H2N atoms, Fig. 6On the N atoms in Fig. 7a). Especially highlighted in Fig. 7b) is a short H7C⋯H10A contact, reflected as a faint-red spot on the dnorm-Hirshfeld surface, with a separation of 2.14 Å, which is 0.26 Å shorter than sum of the van der Waals radii.The amide-N—H⋯O(amide) hydrogen bond, which serves to link cations, is shown as bright-red spots near the amide-O1 and amide-H3W-molecule, the H⋯H and H⋯O/O⋯H contacts contribute 47.6 and 52.4% to the Hirshfeld surface, respectively. On account of the C—H⋯O and N—H⋯O interactions evinced for the O1W-molecule, the contribution of H⋯H and H⋯O/O⋯H contacts differ by ∼2% as compared to the near equal contributions for the O2W-molecule (H⋯H = 49.8%: H⋯O/O⋯H = 50.2%), which does not form analogous contacts.In order to quantify the interatomic contacts for each individual species comprising the asymmetric-unit, the two-dimensional fingerprint plots were also generated. The overall fingerprint plot and those delineated into H⋯H, H⋯O/O⋯H, H⋯C/C⋯H and H⋯N/N⋯H surface contacts are illustrated in Fig. 8de = di ∼ 2.2 Å and de + di ∼ 1.8 Å, respectively, Fig. 8The most significant intermolecular contacts involving the anion are the H⋯H and H⋯O/O⋯H contacts; these contacts tipped at de = di ∼2.2 Å, Fig. 8C⋯H10A contact listed in Table 2de = di ∼1.9 Å in Fig. 8II centre and the carboxylate ligand is reflected in a very low contribution of O⋯Cu/Cu⋯O contacts (0.1%) to the overall Hirshfeld surface of the cation.For the cation, H⋯H contacts contribute 65.2% to the overall Hirshfeld surface with the shortest contact, manifested in the beak-like peak tipped at 2CH2CONH2 arms  Å] occupying the apical position. While the trans-orientated Cu—N(tertiary) bond lengths of 2.083 (4) and 2.086 (4) Å are longer than Cu—N(secondary) bonds of 2.035 (4) and 2.045 (4) Å, the differences between the short and long bond lengths are not as great as noted above for (I)meso isomer, the coordination geometry changes to trigonal-bipyramidal with the amide-O atom occupying an equatorial position, forming a significantly shorter Cu—O bond length [2.007 (4) Å] compared to that in the racemic isomer. The tertiary-N atoms occupy axial positions and form Cu—N(tertiary) bond lengths of 2.063 (4) and 2.088 (4) Å which overlap with the Cu—N(secondary) bond lengths of 2.077 (4) and 2.090 (3) Å. The foregoing demonstrates a dependency of the Cu atom coordination geometry and the magnitudes of putative Cu to O interactions on the nature of the counter-anion and isomeric form of the ligand.There are two relevant structures in the literature available for comparison having closely related 14-membered tetraaza macrocycles bearing two pendent N-bound CHSynthesis ofN-carbamoylethyl pendent derivative (tet-am): The isomeric ligand, tet-a , dissolved in hot methanol (50 ml), and acrylamide , taken in a minimum amount of hot methanol, were mixed. The reaction mixture was refluxed for about 12 h, cooled to room temperature, filtered and allowed to stand for three days to evaporate slowly. The white product that formed, tet-am, was separated by filtration, washed with methanol followed by water and finally dried in a desiccator over silica gel; m.p. 458 K.2CCH3)2·4H2O (I)[Cu(tet-am)] and copper(II) acetate monohydrate  were dissolved separately in hot methanol (25 ml) and mixed while hot, resulting in an immediate colour change. The solution was heated on a steam-bath until the volume was reduced to less than 10 ml. After standing overnight, the sticky material that had formed was dissolved in a minimum amount of ethanol followed by the addition of excess diethylether. The liquid portion was decanted and the remaining violet precipitate, (I)Uiso(H) set to 1.2–1.5Ueq(C). The O- and N-bound H atoms were located in a difference-Fourier map and were refined with O—H = 0.82±0.01 and N—H = 0.86±0.01 Å distance restraints, and with Uiso(H) set to 1.5Ueq(O) and 1.2Ueq(N), respectively.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989021012184/hb8001sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989021012184/hb8001Isup2.hklStructure factors: contains datablock(s) I. DOI: 2122547CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


Crystals suitable for single-crystal X-ray diffraction were obtained by recrystallization from acetone. The aromatic core of the molecule is not planar with the diethylamino group and with the carboxyl group that are rotated out of the 2-oxo-2H-chromene plane by 6.7 (2)° and 11.4 (2)°. The NC2 unit of the diethylamino group is planar with an angle sum close to 360°. Intermolecular Car—H⋯Ocarbonyl interactions lead to the formation of chains parallel to the b axis. X-ray powder diffraction analysis proves that the title compound was obtained as a pure phase.The title compound, C H-1-benzopyran-2-ones are fluorophores with a wide range of biological and chemical applications -2-oxo-2H-chromene-3-carboxylic acid and allyl bromide with potassium carbonate for deprotonation and dry N,N-dimethylformamide as solvent  and 13C NMR  spectroscopy, mass spectrometry, IR spectroscopy and elemental analysis. Recrystallization from acetone led to crystals that were characterized by single-crystal X-ray diffraction. Based on the results of the structure determination, a powder X-ray pattern was calculated and compared with the experimental pattern, revealing that the title compound was obtained as a pure phase .In a current research project, we planed to insert a coumarin moiety as part of a pH-sensitive polymer to visualize material damage. For this purpose, allyl 7-(diethylamino)-2-oxo-2nt Fig. 1. The obt17H19NO4, consists of a central 2-oxo-2H-chromene (2-benzopyrane) unit with a carboxylic acid allyl ester in 3-position and a diethylamino group in 7-position. All atoms of the molecule are in general positions  Å from the least-squares plane calculated through C1–C7 and O1 and O2. The carboxyl group  is slightly twisted from the 2-oxo-2H-chromene unit, with the dihedral angle between the plane calculated through the ring system and that of the carboxyl group being 6.7 (2)°  of the diethylamino group is nearly planar with a maximum deviation of the N atom from the mean plane of 0.0873 Å; planarity is also obvious from the sum of the C—N—C angles of 358.9°. This unit is rotated from the 2-oxo-2H-chromene plane by 11.4 (2)° ° Fig. 3. The NC3)° Fig. 3, which pH-chromene unit and a carbonyl oxygen atom of a neighbouring molecule into chains extending parallel to the crystallographic b axis -diethyl tartrate-2,3-bis(7-diethylaminocoumarin-3-carboxylate) and the second is -N,O-bis(7-diethylaminocoumarin-3-carbonyl)-threonine methyl ester . For the reaction, flasks were flame-dried, evacuated and flooded with a stream of nitrogen. The NMR spectra were measured with a Bruker AvanceNeo 500  in dimethylsulfoxide-d6 (deutero) as solvent. TMS was used as reference. The melting point was measured with a Melting Point Apparatus from Electrothermal. The mass spectrum was measured in the positive mode with an AccuTOF GCV 4G . Rf values were determined by thin-layer chromatography using ALUGRAMM® Xtra Sil G/UV254 plates (Machery-Nagel). Flash column chromatography was performed using cartridge SNAP Ultra 25 g (Biotage®) on a Isolera one (Biotage®). Infrared spectroscopy was performed on a Perkin–Elmer 1600 series FTIR spectrometer. An AG531-G Golden-Gate-Diamond-ATR unit was used. The elemental analysis was performed with a vario MICRO CUBE (Elementar). The probe was put into a zinc container and was burned in an oxygen atmosphere.All reagents and solvents were commercially available and were used without further purification: allyl bromide (abcr), 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid  and potassium carbonate  were suspended in dry N,N-dimethylformamide (20 ml). Allyl bromide  was added and the solution was stirred for 21.5 h at room temperature. After addition of water (50 ml), the mixture was extracted with dichloromethane (4 × 20 ml). The combined organic layer was washed with 1M NaOH solution (30 ml) and dried with magnesium sulfate. After filtration, the solvent was removed in vacuo. The crude product was purified by flash column chromatography on silica gel  to yield the title compound  as a yellow solid. A small amount of the title compound was recrystallized from acetone, leading to crystals suitable for single crystal X-ray diffraction.Under nitrogen atmosphere, 7-(diethylamino)-2-oxo-21H NMR : δ = 8.59 , 7.65 , 6.78 , 6.54 , 6.01 , 5.48–5.22 , 4.72 , 3.48 , 1.14  ppm. 13C NMR : δ = 163.1 , 158.1 , 157.0 , 152.9 , 149.5 , 132.7 , 131.9 , 117.6 , 109.8 , 107.0 , 106.9 , 95.8 , 64.7 , 44.4 , 12.3  ppm. MS : m/z (%) = 301.13 (43) [M]+., 244.10 (20) [M –OCH2CH=CH2]+. HR–MS : found: m/z = 301.1313 [M]+., calculated: m/z = 301.1314 [M]+. (Δ = 0.32 ppm). IR (ATR) wavenumbers: 2972 , 1729, 1685 , 1585 , 1216, 1185, 1114  cm−1. Elemental analysis C17H19NO4 calculated: C: 67.76, H: 6.36, N: 4.65; found: C: 67.67, H: 6.38, N: 4.54.Melting point: 361 K. Uiso(H) = 1.2Ueq(C) (1.5 for methyl H atoms) using a riding model.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021002218/wm5600sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021002218/wm5600Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021002218/wm5600sup3.tifFigure S1: 1H NMR spectrum of 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid allyl ester in dimethylsulfoxide-d6. DOI: Click here for additional data file.10.1107/S2056989021002218/wm5600sup4.tifFigure S2: 13C NMR spectrum of 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid allyl ester in dimethylsulfoxide-d6. DOI: Click here for additional data file.10.1107/S2056989021002218/wm5600sup5.tifFigure S3: Experimental (top) and calculated XRPD pattern (botom) of the title compound measured with copper radiation. DOI: Click here for additional data file.10.1107/S2056989021002218/wm5600Isup6.cmlSupporting information file. DOI: 2064943CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Previous reports implied a possible link between PES1 and lipid metabolism. However, the role of PES1 in regulating T2DM related lipid metabolism and the effect of ketogenic diet (KD) on PES1 have not been reported. The aim of present study is to explore the role of PES1 in effects of KD on diabetic mice and its mediated mechanism.Ay mice were fed with standard diet (SD) and KD, respectively. Simultaneously, McArdle 7777 cells were treated by β-hydroxybutyric acid (β-HB), Pes1 siRNA or Pes1 overexpression plasmid, respectively. Additionally, liver-conditional knockout (CKO) of Pes1 in vivo was applied.Male C57BL/6J and KKPes1, the protein levels of p300, SREBP1c, FASN, SCD1, Caspase1, NLRP3 and GSDMD were dramatically downregulated in livers, and the plasma and hepatic TG, IL-1β and IL-18 were decreased as well. The similar outcomes were also observed in β-HB and Pes1 knockdown treated hepatocytes. By contrast, Pes1 overexpression in cultured hepatocytes showed that these levels were significantly enhanced, which were, however reduced under β-HB treatment. Mechanistically, we discovered that β-HB decreased CHOP binding to the Pes1 promoters, resulting in the downregulation of PES1, thereby reducing PES1 binding to p300 and Caspase1 promoters. The inhibition of p300 and Caspase1 expression elicited the dramatic suppression of acetylation of SREBP1c via its interaction with p300, and the decreased GSDMD levels. Besides, knockdown of Caspase1 also alleviated the TG levels in cultured hepatocytes.Hepatic PES1 expression in diabetic mice was markedly increased, which was suppressed by KD feeding with an accompanying reduction of hepatic and plasma triglycerides (TG). In mice with CKO of KD may improve lipid dysregulation in type 2 diabetic mice by downregulating hepatic PES1 expression.The online version contains supplementary material available at 10.1186/s10020-021-00429-6. Type 2 diabetes mellitus (T2DM), characterized by insulin resistance (IR) and pancreatic β-cell dysfunction, is a heterogeneous metabolic disorder , has been proposed to attain a remarkable decline of hyperglycemia in T2DM Abbasi . KD is tCurrently, we unexpectedly observed that hepatic pescadillo 1 (PES1) expression in T2DM mice were markedly elevated. PES1, also known as pescadillo ribosomal biogenesis factor 1, or NOP7, or YPH1, was originally found in zebrafish embryos  and Pes1 overexpression plasmid were purchased from GENERAL BIOL .β-HB (166898) was bought from Sigma Chemical Co . Triglycerides (TG), total cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and β-HB assay kits were from Nanjing Jiancheng Bioengineering Institute . The ELISA kit for insulin was obtained from Jianglai Corp . Flag-tag (20543-1-AP), β-actin (66009-1-Ig) and Caspase1 (22915-1-AP) antibodies for western bolting, and PES1 (13553-1-AP) antibody for immunofluorescence were obtained from Proteintech, Chicago, USA. PES-1 (NBP2-55211) antibody for western bolting and SREBP1c antibody (NB600-582) for immunofluorescence were bought from Novus Biologicals. SREBP1c (191857), SCD1 (ab19862) and SREBP2 (ab155017) antibodies for western bolting were purchased from Abcam. FASN (3180S) antibody for western bolting was from Cell Signaling Technology . NLRP3 (DF7438) and GSDMD (AF4012) antibodies for western bolting were from Affinity. CHOP (sc-7351), p300 (sc-48343) and Ac-SREBP1c  antibodies were bought from Santa Cruz Biotechnology. RNAex Pro Reagent (AG21102), reverse transcription kits (AG11707), SYBR Green qPCR SuperMix (AG11718) were bought from Accurate Biology. Cell counting kit-8 (CCK-8) (C0005) was from Target Mol . Chromatin immunoprecipitation (ChIP) assay kit (P2078) was purchased from Beyotime. Rat Ay (KK.Cg-Ay/J) mice were from Beijing Vital River. The housing unit was maintained at constant temperature 22–25 °C and 50–60% relative humidity with a 12/12-h light/dark cycle and free access to tap water. KKAy mice were fed with commercial high-fat diet  ad libitum for 4 weeks to increase their plasma glucose levels. After 4 weeks, all mice were divided into four groups (10–12 mice per group), including C57BL/6J fed with standard diet  (C57BL/6J-SD) and ketogenic diet  (C57BL/6J-KD), and KKAy fed with SD (KKAy-SD) and KD (KKAy-KD). The composition of the diet was listed in Additional file Five-week-old male C57BL/6J and KKFasting glucose was measured weekly by Roche blood glucose meter. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed before the mice were sacrificed, the concentrations of glucose and insulin for intraperitoneal injection were 2 mg/g body weight and 0.7 mU/g body weight, respectively. Blood glucose samples were tested at 0, 30, 60, 90, 120, 150 min and 0, 20, 40, 60, 80, 100, 120 min, respectively. Area-under-curve (AUC) was calculated by the trapezoid rule.By the end of experiment, all fasted mice were euthanized with 10% chloral hydrate and then killed by cervical dislocation to obtain tissues and blood samples. Blood was immediately centrifuged at 3000 rpm for 10 min and the levels of insulin in plasma were detected by enzyme linked immunosorbent assay (ELISA) kits. The homeostatic model assessment of insulin resistance (HOMA-IR) index was calculated using the following formula: fasting insulin levels (mIU/L) × fasting glucose levels (mmol/L)/22.5. The remaining plasma was stored at − 80 °C for later analysis. The liver tissues were rinsed with cold phosphate-buffered saline (PBS). Small portions of liver tissues were fixed in 4% paraformaldehyde solution for oil red O and hematoxylin–eosin (H&E) staining respectively. Then two-third of liver per mice was frozen in liquid nitrogen immediately and kept at − 80 °C for immunoblotting and biochemical analysis. The remaining livers were preserved in RNAlater for qRT-PCR test. TC, TG, ALT and AST were determined using enzymatic kits in accordance with the manufacturer's instruction.2 at 37 ℃. The concentration and time of β-hydroxybutyric acid (β-HB) treatment were selected depending on cell counting kit-8 (CCK-8) test. Specifically, McArdle liver cells were seeded in the 96-well plates for 24 h. Then by the β-HB treatment with a series of concentrations  and times , CCK-8 reagents (10 µL) were added into the treated cells. Next, the cells were incubated for 1–4 h at 37 °C. Simultaneously, the medium was treated in the same way. The absorbance at 450 nm was detected by the microplate reader. Cellular viability was calculated by the absorbance values, by which the most suitable treatment concentration and time of β-HB were determined.McArdle 7777 rat hepatoma cells were obtained from the American Type Culture Collection . McArdle 7777 cells were cultured in DMEM with high glucose concentration (25 mmol/L), supplemented with 10% FBS and 1% penicillin/streptomycin in an incubation chamber with 5% CO−△△Ct).The total RNAs were extracted using RNAex Pro reagent. The genomic DNA was removed using RNase-free DNase. Purified total RNA 1 μg) was reverse-transcribed using reverse transcriptase. qRT-PCR was performed with a Roche Light Cycler 480 System with SYBR Green Super Mix using gene-specific primers  were homogenized in lysis buffer, respectively. Total lysates (20 μg per well) were separated electrophoretically by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were respectively incubated for 24 h with CHOP, PES1, p300, SREBP1c, SREBP2, FASN, SCD1, NLRP3, Caspase 1, GSDMD antibodies at 4 °C. β-Actin was used as a loading control. After being washed 8 min for 4 times with TBST buffer, the membranes were incubated with secondary antibodies for 60 min. The enhanced chemiluminescence reagent was used. The signal was then detected by the digital imaging equipment.McArdle 7777 cells were seeded onto 12-well plate containing cell climbing slides for 24 h. After β-HB treatments, cells were washed thrice with PBS and fixed with 4% paraformaldehyde for 20 min. Nonspecific binding sites were blocked with 5% normal bovine serum in PBS. The 12-well plates were incubated with the mixture of primary antibodies including PES1 (1:250) and SREBP1c (1:250) at 4 °C overnight. After PBS washing, the 12-well plates were incubated for 60 min with the Alexa Fluor 488 conjugated secondary antibody  or Alexa Fluor 568 conjugated secondary antibody . Cell climbing slides were taken out from the 12-well plate and buckled upside down on the glass slide containing DAPI. All sections were mounted and observed by a confocal microscope .Pes1 and Caspase1 small interfering RNA (siRNA) and Lipofectamine 3000  were mixed in serum-free medium for 20 min, respectively. Then the mixture was added to the culture medium to transfect McArdle 7777 cells. After 6 h, the cells continue to be cultured in fresh medium for 48 h. Finally, cells were collected for subsequent measuring TG, TC, and running Western Blotting and qRT-PCR. The sequences of Pes1 siRNA were 5ʹ-UGAAGAAGCGAGAGAAGUATTT-3ʹ (forward) and 5ʹ-UACUUCUCUCGC UUCUUCATT-3ʹ (reverse). The sequences of Caspase1 siRNA were 5ʹ-AGGAAGA GAUGGAUACAAUTT-3ʹ (forward) and 5ʹ-AUUGUAUCCAUCUCUCUUCCUTT-3ʹ (reverse). The scrambled siRNA control sequences were 5ʹ-UUCUCCGAA CGUGUCACGUTT-3ʹ (forward) and 5ʹ-ACGUGACACGUUCGGAGAATT-3ʹ (reverse).Rat Pes1-flag and Lipofectamine 3000 were mixed in serum-free medium for 20 min. Then the mixture was added into the culture medium to transfect McArdle 7777 cells for 6 h. After being cultured in fresh medium for 24 h, the cells were divided into three groups for different treatments, including negative control (without transfection of Pes1 plasmid or any reagent), Pes1-plasmid and Pes1-plasmid plus β-HB. After being cultured for 24 h, cells were collected for subsequent assays of TG, TC, Western Blotting and qRT-PCR.The purified plasmid of Pes1 promoter, PES1 binding to the p300 and Caspase1 promoters were analyzed by chromatin immunoprecipitation (ChIP). Briefly, cells were crosslinked with 1% formaldehyde for 10 min, neutralized with 125 mM glycine pH 2.5 and washed in PBS. Nuclei were prepared by hypotonic lysis buffer  and centrifugation, and resuspended in SDS lysis buffer, and incubated on ice for 10 min to be fully lysed. Chromatin was sonicated with bioruptor (Diagenode) to be 200–1000 bp average fragment size and cleared by centrifugation. The anti-CHOP, anti-PES1 antibodies or control rabbit IgG  was respectively added into an aliquot of 200 μL sonicated lysate, and then 20 μL washed protein A/G-agarose beads  was added. The mixture was rotated at 4 °C for 2 h, and then centrifuged at 1000g for 1 min at 4 °C to wash the beads. The washed beads were resuspended in TE buffer, vortexed 10 s and boiled for 10 min. The samples and sonicated lysates were treated with 1 μL of 20 mg/mL proteinase K. After being centrifuged at 12,000g for 5 min at 4 °C, the digested DNA was used for qRT-PCR assay. Primers for the CHOP binding site in Pes1 promoter, the PES1 binding site in the rat p300 and Caspase1 promoters were as follows: CHOP-Pes1 binding site (forward) 5ʹ-CTGGTACGTGGGTGCAGTTTGG-3ʹ, CHOP-Pes1 binding site (reverse) 5ʹ-CACACAGGGATGAACATAAGTGAGAGG-3ʹ; PES1-p300 binding site (forward) 5′-TCCTCTTGCTGTCTGACTTGTTTGAG-3′, PES1-p300 binding site (reverse) 5′-AAGATGTTGAGCCTGTTCTCTGAGTTC-3′; PES1-Caspase1 binding site (forward) 5′-GGAGCAGGGAAACGATGTATGT GAG-3′, PES1-Caspase1 binding site (reverse) 5′- TTGCCCTCAGGATCTTGTCTG TTTAAG-3′.The binding of CHOP (C/EBP-homologous protein) to the Every extracted protein stock solution from cultured cells or liver tissues was divided into two aliquots. The small part was directly used for input assay, and the remaining big portion was pre-cleared with protein A/G-agarose  for 2 h. The supernatant after pre-clearance was collected by centrifugation, and further divided into two equal portions. Ac-SREBP1c (acetylated SREBP-1c), p300 and nonimmune IgG  antibodies were respectively added in the supernatant (2 μg/per portion) and incubated at 4 °C overnight in rotating equipment. After centrifugation, the precipitates were collected and washed with cold lysis buffer for six times. The mixture of precipitates and loading buffer was boiled and then Ac-SREBP1c, p300, SREBP1c levels were detected using immunoblotting.Pes1 CKO mice were generated by collaboration with Nanjing Institute of Biomedicine, Nanjing University . Positive F1 generation mice were achieved by CRISPR–Cas9 technology. And hepatic Pes1 CKO mice were obtained by positive F1 generation crossing with ALB-Cre mice. Genotyping was performed by PCR using genomic DNA extracted from murine tails at 3–4 weeks. The mice were classified into wild-type littermates  and Pes1(−/−)  relying on genotype. The primer sequences for the transgenic Cre mice were as follows: Cre sense, 5ʹ-TTGGCCCCTTACCATAACTG-3ʹ; Cre antisense, 5ʹ-GAAGCAGAAG CTTAGGAAGATGG-3ʹ. The primer sequences for genotyping the Pes1 alleles were as follows: shared sense, 5ʹ-TTCCTCACCCTCAGCATTAGʹ; wild-type antisense, 5ʹ-GAGATAGACTGCAAGGCACTGT-3ʹ. Only male mice were used for all analyses (10–15 mice per group). After 16 weeks, mice were sacrificed after anesthetization to obtain the serum and livers for consecutive measuring TG, TC, and running Western Blotting and qRT-PCR.The parent of P < 0.05 was considered statistically significant. The data were analyzed using SPSS version 22.0 . Graphics were constructed using GraphPad Prism 7 .Normally distributed data were expressed as mean ± SEMs. The continuous variables were analyzed either by Student’s t test, or by ANOVA followed by Student–Newman–Keuls q test for multiple comparisons. Ay-KD group showed no significant difference from those in the KKAy-SD groups, the similar results were also observed in the C57BL6J mice  was found  in the murine livers , treatment times . b, c Protein levels of PES1 in hepatocytes were detected in different concentrations and times of β-HB treatment. Data were shown as mean ± SEM for each experiment performed independently 3 times. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control . Figure S2. Hepatic Pes1 gene transcription in CKO mice was detected by qRT-PCR. Data were shown as mean ± SEM for each experiment performed independently 3 times. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control (Student’s t test). Figure S3. KD enhanced the fatty acid β-oxidation in normal and diabetic mice. a, b The protein levels of hepatic CPT1 and HMGCS2 were detected by Immunoblotting. Values are means ± SEM for each experiment performed independently 3 times. SD (Standard diet), KD (Ketogenic diet). *P < 0.05, **P < 0.01, ***P < 0.001 compared with control for C57BL/6J, #P < 0.05, ##P < 0.01, ###P < 0.001 compared with control for KKAy, +P < 0.05, ++P < 0.01, +++P < 0.001 compared with C57BL/6J . Figure S4. The ratios of β-HB levels in the C57BL/6J and KKAy mice fed with KD and SD. Values are means ± SEM for each experiment performed independently 3 times. SD (Standard diet), KD (Ketogenic diet).


The first error regards Table 3, which was published with a wrong title. The correct title is “iK concn (mM) ± SDa.” Second, in the originally published Volume 12, no. 6, e02206-21, 2021,  F1 Crossref missing


Recently, quasi-two-dimensional (Q-2D) perovskites have received much attention due to their excellent photophysical properties. Phase compositions in Q-2D perovskites have obvious effect on the device performance. Here, efficient green perovskite light-emitting diodes (PeLEDs) were fabricated by employing o-fluorophenylethylammonium bromide (o-F-PEABr) and 2-aminoethanol hydrobromide (EOABr) as the mix-interlayer ligands. Phase compositions are rationally optimized through composition and interlayer engineering. Meanwhile, non-radiative recombination is greatly suppressed by the introduction of mix-interlayer ligands. Thus, green PeLEDs with a peak photoluminescence quantum yield (PLQY) of 81.4%, a narrow full width at half maximum (FWHM) of 19 nm, a maximum current efficiency (CE) of 27.7 cd/A, and a maximum external quantum efficiency (EQE) of 10.4% were realized. The results are expected to offer a feasible method to realize high-efficiency PeLEDs. Metal-halogen perovskites are emerging as potential candidates for light-emitting diodes due to their high color purity , high photoluminescence quantum yield (PLQY), facile adjustable photoelectronic properties, and solution processability . ImpressThree-dimensional (3D) perovskites with continuous octahedral frameworks generally feature in a weak exciton binding energy . Exciton−1, and a maximum EQE of 10.4%. The results demonstrated here offer a simple path to realize efficient PeLEDs.In this work, o-F-PEABr and EOABr were employed as the mix-interlayer ligands to fabricate Q-2D PeLEDs. Phase compositions and energy levels can be effectively regulated by the o-F-PEABr ligand. On combining with the EOABr ligand, the non-radiative recombination channels in these Q-2D perovskites are greatly suppressed. Efficient green PeLEDs based on mix-interlayers were realized with an emission peak at 509 nm, a maximum CE of 27.7 cd A2Csm-1PbmBr3m+1 with various m values . Perovskite emitters were prepared by the one-step spin-coating method. To study the effect of phase compositions on PeLED performance, a group of devices with a configuration of ITO (100 nm)/polyvinylcarbazole (PVK):N,N′-bis(4-butylphenyl)-N,N-bis(phenyl)-benzidine (TPD) (30 nm)/perovskite emitters (60 nm)/3,5-Tris(1-phenyl-1H-benzimidazol-2-yl)benzene (TPBI) (35 nm)/LiF (1 nm)/Al (150 nm) were fabricated. PVK:TPD acted as a hole-transporting layer, and TPBI was used as an electron-transporting layer. The perovskite emitters with m = 1, 2, 3, 4 were employed as the light-emitting layers. PeLEDs based on the perovskite emitters with m = 1 were also fabricated; however, device performance could be hardly observed. As previously reported, this should be attributed to their poor film morphology, large hole-injection barrier, and serious traps assisting non-radiative recombination  PeLEDs show optimum device performance with a peak luminance of 7,290 cd/m2, a maximum CE of 14.2 cd A−1 (on) at 3.0 V certifies the carriers were effectively injected into the perovskite emitters (The electroluminescence (EL) performance of these PeLEDs is shown in 2 cd A−1 , and a m2 cd A−1 . The turemitters , which cemitters . Therefo2Csm-1PbmBr3m+1 Q-2D perovskite can be effectively regulated by changing the m values. The single absorption peak at 396 nm of m = 1 (o-F-PEA2PbBr4) perovskite is attributed to the n = 1 phase. The absorption peaks of m = 2 (o-F-PEA2CsPb2Br7) perovskite at 396, 428, and 456 nm are attributed to n = 1, 2, 3 phases, respectively. The n = 1, 2 phases could be greatly inhibited as the m = 3 (o-F-PEA2Cs2Pb3Br10) and 4 (o-F-PEA2Cs3Pb4Br13) perovskites. The PLQY of m = 1, 2, 3, 4 is 1.1%, 6.8%, 58.7.1, and 46.8%, respectively.As the ultraviolet–visible (UV–Vis) absorption spectra shown in The morphology of these perovskites was studied through the scanning electron microscopy (SEM) test. As shown in x o-F-PEA y)2Cs2Pb3Br10 (x + y = 1). The phase compositions were further engineered by tuning the mix-interlayer ligand molar ratio with x:y = 1:8, 2:8 toward efficient quasi-2D perovskites. UV–Vis absorption and photoluminescence (PL) spectra of these samples are exhibited in The employment of mix-interlayer ligands with synergistic effects is an effective way to improve the device performance. Here, mix-interlayer perovskites were fabricated by introducing the EOABr ligand with the general formula of (EOA ave) of 57.6 ns, which is larger than that of m = 3 (47.1 ns) perovskites, demonstrating that excitons in the mix-interlayer perovskites can live for a longer time. r) and non-radiative (knr) transition rates. The m = 3 perovskite shows a moderate kr:knr ratio of 1.43. The 1:8 and 2:8 perovskites show significantly increased kr:knr ratios of 4.36 and 3.78, respectively. Therefore, non-radiative channels in mix-interlayer Q-2D perovskites are suppressed through more effective interlayer ligand passivation.PLQYs of these samples are shown in 3 (stand PDF# 18-0364) was also included for comparison. The 2θ diffraction peaks at 15.24° and 30.71° are observed from all the perovskite films, which ascribe to the (100) and (200) planes of the large-n phase (n ≥ 5) perovskite similar to CsPbBr3. The stronger XRD peak intensity of 1:8 and 2:8 samples indicated more dominant crystal orientation.The X-ray diffraction (XRD) test was carried out to analyze the crystal structures of these mix-interlayer perovskites . The difx o-F-PEA y)2Cs2Pb3Br10 can restrain leakage current and facilitate the realization of highly efficient PeLEDs.The film morphology of these samples was examined using SEM . The SEMEncouraged by the excellent optical and physical properties, the performance of these mix-interlayer perovskites was tested with the same device configuration of ITO (100 nm)/PVK:TPD (30 nm)/perovskite emitters (60 nm)/TPBI (35 nm)/LiF (1 nm)/Al (150 nm). on of mix-interlayer perovskites comes from the slightly reduced hole carrier injection barrier.The energy levels of perovskite emitters are exhibited in −1 and 10.4%, respectively. The improved EQE can be explained by the higher PLQY, larger kr:knr, and better film quality owing to the mix-interlayer ligands.All the EL performance parameters are summarized in 2. The half-lifetime (T50) amounts to the time it takes to decrease the luminance to 50% of its initial value. As shown in 50 lifetime of the m = 3, 1:8, and 2:8 PeLEDs is 34.6, 48.5, and 7.2 min, respectively. The good device stability of 1:8 PeLEDs is probably due to the better film quality, matched energy levels, and efficient carrier injection. Statistical EQE performance of the 1:8 perovskite for 30 devices is shown in The device stability is one of the key parameters of PeLEDs. The spectra stability of 1:8 PeLEDs was first studied to investigate the device stability. As shown in 50 lifetime of 48.5 min. This work can provide a simple and feasible strategy for improving the performance of Q-2D PeLEDs.In summary, efficient green PeLEDs have been realized by introducing o-F-PEABr and EOABr as the mix-interlayer ligands. The phase compositions, defect passivation, and film quality can be easily adjusted through composition and mix-interlayer engineering. Under the optimization of optical and electric properties, efficient green PeLEDs based on 1:8 mix-interlayers were obtained with a peak CE of 27.7 cd/A, a maximum EQE of 10.4%, and a TMaterials. Cesium bromide , lead bromide , o-F-PEABr (99.5%), TPD (99.5%), and EOABr (99.5%) were purchased from Xi’an p-OLED Corp. Dimethyl sulfoxide  was purchased from Acros, PVK (average Mw ∼1100000) was purchased from Sigma-Aldrich, and TPBI (99.5%) was purchased from Luminescence Technology Corp. All materials were used as received without further purification.2 in the molar ratios of 2:0:1, 2:2.4:2, 2:3.6:3, and 2:4.8:4 in DMSO under continuous stirring for 6 h at 50°C, respectively. The mix-interlayer Q-2D perovskite precursor solution of 1:8 and 2:8 was prepared by dissolving o-F-PEABr, EOABr, CsBr, and PbBr2 in the molar ratios of 1.78:0.22:3.6:3 and 1.6:0.4:3.6:3 in DMSO under continuous stirring for 6 h at 50°C, respectively. The Pb2+ concentration in perovskite precursor solution is 0.15 M.The o-F-PEABr Q-2D perovskite precursor solution of m = 1, 2, 3, and 4 was prepared by dissolving o-F-PEABr, CsBr, and PbBr3 for 30 min and transferred into a nitrogen-filled glove box. 30 nm of PVK:TPD  was spin-coated and then baked at 130°C for 20 min. 1,4-Dioxane was spin-coated onto PVK:TPD at 3,000 rpm to increase the surface wettability. Then, perovskite emitters were spin-coated from the precursor solution and annealed at 95oC for 20 s. Finally, TPBI (35 nm), LiF (1.0 nm), and Al (150 nm) were evaporated under a pressure of 1 × 10–4 Pa. The thickness of the evaporated materials was monitored by a quartz crystal thickness monitor . Deposition rates of TPBI, LiF, and Al were 1.5 Å s−1, 0.1 Å s−1, and 4 Å s−1, respectively. The emission area of these PeLEDs was 2 mm * 2 mm. All the fabrication processes were accomplished inside a nitrogen dry box. The oxygen and moisture of nitrogen dry box were less than 1 ppm. Current density (J)–voltage (V) curves were measured by using a dual-channel Keithley 2400 instrument. The EL spectra, CE, and EQEs were measured by using an integrating sphere, a multi-channel analyzer PMA-12, and an external quantum efficiency measurement system   . Before UV–Vis absorption spectra were collected by a SHIMADZU/UV-3600 PLUS spectrophotometer. XRD spectra were measured by a multipurpose X’Pert PRO system. SEM images were taken with a ZEISS/SIGMA500 system. PL spectra were measured using an Edinburgh FL980 fluorescence spectrophotometer with a 375 nm xenon lamp as the excitation light source. Time-resolved PL decay spectra were measured with an Edinburgh FL980 fluorescence spectrophotometer with a 371.6 nm ps diode laser as the excitation light source. PLQYs of the perovskite films were measured by a commercialized PLQY measurement system from Ocean Optics with a 375 nm LED as the excitation light source. The energy level values were measured by atmospheric ultraviolet photoelectron spectroscopy (Riken Keiki AC-3).


After publication, the authors became aware that the sequences of pE30-2 were derived from the PhiX sequencing control. We apologize for the inconvenience caused.Volume 10, no. 45, e00907-21, 2021, Page 1, abstract: “two circular chromosomes and two plasmids” should read “two circular chromosomes and one plasmid.”Page 2, Table 1: line 4 should be deleted. The total size (bp) should read “4,304,943,” and the total number of coding sequences should read “3,902.”Page 2, line 2: “two plasmids ” should read “one plasmid .”Page 2, lines 4 and 5: “Plasmids pE30-1 and pE30-2 contain 43 and 6 protein-coding genes, respectively.” should read “Plasmid pE30-1 contains 43 protein-coding genes.”AP025028, AP025029, AP025030, and AP025031” should read “accession numbers AP025028, AP025029, and AP025030.”Page 2, line 10: “accession numbers


Scientific Reports 10.1038/s41598-021-94819-9, published online 28 July 2021Correction to: 2]”, TS “[N/mm2]”, and “Impact toughness [J] at 0 °C” were incorrect. The incorrect and correct values appear below.The original version of this article contained an error in Table 3, where the values for “YS [N/mmIncorrect:Correct:The original Article has been corrected.


Contents listing of the journal.Please provide a synopsis (of no more than two sentences) for inclusion in the  N}copper(I)), [Cu2Cl2(C8H9NS)2], and di-μ-bromido-bis-(prop-2-en-1-yl)sulfanyl]pyridine-κN}copper(I)), [Cu2Br2(C8H9NS)2], were obtained by alternating-current electrochemical synthesis starting from an ethanolic solution of 2-[(prop-2-en-1-yl)sulfanyl]pyridine (Psup) and the copper(II) halide. The isostructural crystals are built up from centrosymmetric [Cu2Hal2(Psup)2] dimers, which are formed due to the π,σ-chelating behavior of the organic ligand. In the crystals, the dimers are linked by C—H⋯Hal hydrogen bonds as well as by aromatic π–π stacking interactions into a three-dimensional network.The title compounds, di-μ-chlorido-bis-(prop-2-en-1-yl)sulfanyl]pyridine-κ For instance, the first examples of Cu(C6H5SO3), Cu(p-CH3C6H4SO3) or CuHSO4 π-complexes as well as the direct CuI⋯F(SiF62–) interaction have been observed in copper(I) π-compounds with allyl derivatives of triazole and thiadiazole 2] (I) & [Cu2Br2(Psup)2] (II) with 2-[(prop-2-en-1-yl)sulfanyl]pyridine (Psup), C8H9NS.Over the last two decades, increased interest has also been devoted to the crystal engineering of copper(I)–olefin complexes with allyl derivatives of heterocyclic compounds  ion and one halide ion in the asymmetric unit. As shown in Figs. 12Hal2(Psup)2] [Hal = Cl (I) or Br (II)] dimers, which are formed due to the chelating behavior of the organic ligand. A close to trigonal–pyramidal coordination environment of the CuI cation includes the η2 allylic C=C bond, the pyridine N atom and a Hal1 ion in the basal plane  −x + 1, −y + 1, −z + 1) ion at 2.6186 (9) Å in I and at 2.7113 (6) Å in II. The corresponding four-coordinate geometry indices τ4  and 0.83 (II). For comparison, in the structures of previously studied CuCl and CuBr π,σ-complexes with allylacetoneoxime, the Cu—Halap distances are slightly higher at 2.719 (5) and 2.778 (4) Å  Å (I) and to 1.354 (6) Å (II) in comparison with an uncoordinated allylic C=C bond  and (II) has synclinal conformation relative to the S1—C7 bond and an antiperiplanar conformation relative to the C7—C8 bond .Being π-connected to the metal center, the C8=C9 bond of the ligand is elongated due to back-donation from an occupied 3I) and (II) features several weak intermolecular interactions. The hydrogen atom H6 of the pyridine ring participates in an intramolecular C—H⋯Hal bond with the Hal ion of the inorganic subunit. The other hydrogen atom H6 of the pyridine ring and the methylene hydrogen atom H7B of the allylsulfanyl substituent are involved in intermolecular C—H⋯Hal bonding, linking the dimeric moieties into a three-dimensional network. The pyridine rings of adjacent dimers are also involved in face-to-face π–π stacking interactions with a centroid–centroid separation of 3.680 (4) Å in I and 3.693 (4) Å in II. The unit-cell packing for (I) is shown in Fig. 4As shown in Fig. 32Hal2} dimer in which a π,σ-chelating ligand is bound to copper(I) are: di-μ-chlorobis[copper(I)] (III) [Cambridge Structural Database -bisdicopper(I) (IV) -bisdicopper(I) (V) benzimidazole-N)copper(I)] (VI) [(2-pyridyl)dimethylsilane]copper} (VII) . Structures (III), (IV), (V) and (VI) are built up from centrosymmetric [Cu2Hal2(Ligand)2] dimers. In the compounds bis[(μ2-chloro)chlorocopper(I)] (XIII) (BEBFOE) and bis[(μ2-chloro)bromocopper(I)] (IX) bis(μ2-chloro)bisdichlorotetracopper(I)] (X)  and (VII) crystallize in the triclinic crystal system in space group et al., 2018aPsup) and the copper(II) halide. For this, a solution of Psup  in 2.0 ml of 96% ethanol was added to a solution of CuCl2·2H2O  or CuBr2  in 3.0 ml of 96% ethanol. The mixture was carefully stirred and then was placed into a small 5.5 ml test tube. A copper wire was wrapped into a spiral of 1 cm diameter. A straight copper wire was placed inside the spiral. These copper electrodes were inserted in a cork and immersed in the aforementioned mixture. The mixture was subjected to alternating current reduction  and after 3–4 days, good-quality slightly yellowish crystals of the title compounds appeared on the copper wire electrodes. Compound I: yield 12%, m.p. 413 K; compound II: yield 8%, m.p. 407 K.Crystals of the title compounds were obtained under conditions of alternating-current electrochemical synthesis (Slyvka Uiso(H) = 1.2Ueq(C) was applied in all cases.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989021011002/hb7993sup1.cifCrystal structure: contains datablock(s) I, II, publication_text. DOI: 10.1107/S2056989021011002/hb7993Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989021011002/hb7993IIsup3.hklStructure factors: contains datablock(s) II. DOI: 2116969, 2116968CCDC references:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


Phellodendron amurense and Coptis chinensis. However, the effects of berberine on double-stranded RNA (dsRNA)-induced macrophages have not been fully reported. In this study, we examined the anti-inflammatory effects of berberine on dsRNA -induced macrophages. Levels of nitric oxide (NO), Prostaglandin E2 (PGE2), first apoptosis signal receptor , cytokines, intracellular calcium, phosphorylated I-kappa-B-alpha (IkB-α), phosphorylated p38 mitogen-activated protein kinase (MAPK), phosphorylated ERK1/2, phosphorylated signal transducer and activated transcription 3 (STAT3), and mRNA expression of inflammatory genes in poly I:C-induced RAW 264.7 mouse macrophages were evaluated. Berberine significantly inhibited the production of NO, PGE2, Fas, GM-CSF, LIF, LIX, RANTES, and MIP-2 as well as calcium release in poly I:C-induced RAW 264.7 cells at concentrations of up to 50 μM. Berberine also significantly inhibited the phosphorylation of p38 MAPK, ERK1/2, IkB-α, and STAT3 in poly I:C-induced RAW 264.7 cells. Additionally, berberine significantly decreased the mRNA expressions of Chop (GADD153), Stat1, Stat3, and Fas in poly I:C-induced RAW 264.7 cells. Taken together, berberine has anti-inflammatory properties related to its inhibition of NO, PGE2, Fas, GM-CSF, LIF, LIX, RANTES, and MIP-2 in dsRNA-induced macrophages via the endoplasmic reticulum stress-related calcium-CHOP/STAT pathway.Berberine is a well-known quaternary ammonium salt that is usually found in the roots of such plants as  Jiang et al. reported that berberine suppresses IL-1β secretion that was induced by the activation of the NLRP3 inflammasome in macrophages2.Berberine , cytokines, chemokines, and prostaglandins. Cytokines, including chemokines and growth factors, also play an important role in hypersensitive reactions against both exogenous and endogenous antigens. Among immune cells, macrophages play a major role in defending the human body against infectious pathogens. Of course, macrophages produce many kinds of inflammatory mediators, including NO, Prostaglandin E2 (PGE2), first apoptosis signal receptor , cytokines, chemokines, growth factors, and prostaglandins, against invasive bacteria, fungi, and viruses.3.In these days, severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) infection has caused global disruption. SARS‑CoV‑2 is thought to bring about hyper-inflammation and cytokine storm, which could be led to multiple organic failures. Thus, it is meaningful to investigate inhibitory effects of natural products on viral infection-associated inflammation. Interestingly, Kim et al. reported in 2008 that in vitro coronavirus replications are inhibited by Coptidis rhizoma and Phellodendron cortex, which are well known to contain berberineStat1), Stat3, C/EBP-homologous protein , and Fas in RAW 264.7 cells as well as phosphorylation of p38 mitogen-activated protein kinase (MAPK), ERK1/2, STAT3, and I-kappa-B-alpha (IkB-α).During viral infections, many double-stranded RNAs are produced by viral replication, which causes viral inflammation. In this study, polyinosinic–polycytidylic acid (poly I:C), a synthetic analog of double-stranded RNA (dsRNA), was used to provoke a hyper-inflammatory reaction in RAW 264.7 mouse macrophages, producing various inflammatory mediators, such as NO, PGE2, Fas, granulocyte macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), lipopolysaccharide-induced CXC chemokine , chemokine ligand 5 , macrophage inflammatory protein (MIP)-1α, and MIP-2, as well as releasing intracellular calcium. Additionally, poly I:C increased mRNA expression levels of signal transducer and activated transcription 1  stress-related calcium-CHOP/STAT pathway.In the previous reports, we showed that bioactive compounds, such as wogonin, oroxylin A, quercetin, and baicalein, exert anti-inflammatory effects on RAW 264.7 cells induced by poly I:CIn this study, the cell viability in RAW 264.7 incubated with poly I:C (50 µg/mL) for 24 h was 39.37 ± 7.82% of the normal value. In the addition, cell viabilities in poly I:C-induced RAW 264.7 incubated with berberine at concentrations of 10, 25, and 50 µM were 164.17 ± 35.34%, 171.2 ± 42.97%, and 183.99 ± 41.35% of the control (poly I:C only) value  plus poly I:C, berberine (25 µM) plus poly I:C, berberine (50 µM) plus poly I:C, and indomethacin (0.5 µM) plus poly I:C for 24 h was 11.67 ± 5.51 pg/mL, 111.33 ± 15.95 pg/mL, 40.5 ± 8.05 pg/mL, 45 ± 18.36 pg/mL, 76.17 ± 12.55 pg/mL, and 8.17 ± 0.58 pg/mL, respectively;Was 13.83 ± 2.47 pg/mL, 3882 ± 32.22 pg/mL, 2823.5 ± 271.99 pg/mL, 2663.33 ± 544.29 pg/mL, 3247 ± 258.45 pg/mL, and 101.33 ± 36.36 pg/mL;Was 186.17 ± 30.19 pg/mL, 2662.33 ± 54.82 pg/mL, 2263.83 ± 173.57 pg/mL, 2201.83 ± 87.51 pg/mL, 2510.17 ± 58.06 pg/mL, and 396.33 ± 26.17 pg/mL;Was 37.33 ± 18.61 pg/mL, 15,174.33 ± 791.08 pg/mL, 12,504.67 ± 444.89 pg/mL, 10,301.67 ± 2223 pg/mL, 13,804 ± 499.75 pg/mL, and 3922.167 ± 197.44 pg/mL;Was 380.17 ± 218.45 pg/mL, 17,803.67 ± 4642.22 pg/mL, 10,973.88 ± 253.51 pg/mL, 10,138.25 ± 1231.07 pg/mL, 11,357.63 ± 813.8 pg/mL, and 11,719.67 ± 874.66 pg/mL;Was 1518.33 ± 2465.6 pg/mL, 25,364.13 ± 908.61 pg/mL, 23,971.17 ± 185.4 pg/mL, 23,135 ± 1122.51 pg/mL, 24,299.63 ± 403.08 pg/mL, and 12,522.5 ± 1143.2 pg/mL;These data indicated that berberine might alleviate hyper-inflammatory reaction leading to excessive production of chemokines and growth factors in macrophages stimulated by poly I:C.Stat1, Stat3, Chop, and Fas. These results mean that berberine inhibits the inflammatory reaction in poly I:C-induced macrophages via the ER stress-related CHOP pathway. The effects were as follows: the mRNA expression of Stat1 in poly I:C-induced RAW 264.7 incubated with berberine at concentrations of 10, 25, and 50 µM for 18 h was 67.74 ± 10.6%, 62.61 ± 7.5%, and 56.98 ± 4.81% of the group treated with poly I:C only, respectively; the mRNA expression of Stat3 was 11.05 ± 0.99%, 12.41 ± 1.1%, and 7.06 ± 0.7, respectively; the mRNA expression of Chop was 32.96 ± 5.56%, 57.71 ± 6.24%, and 51.46 ± 5.9, respectively; the mRNA expression of Fas was 48.44 ± 36.12%, 34.87 ± 5.11%, and 15.95 ± 7.42, respectively.Figure We found that berberine significantly inhibited the phosphorylation of p38 MAPK, ERK 1/2, STAT3, and IkB-α in poly I:C-induced RAW 264.7 cells Fig. . In detaData represented that berberine significantly decreases the levels of Fas and PGE2 in poly I:C-stimulated RAW 264.7 cells , Berberis vulgaris (barberry), Berberis aristata (tree turmeric), Mahonia aquifolium (Oregon grape), Hydrastis Canadensis , Xanthorhiza simplicissima (yellow root), Coptis chinensis (Chinese goldthread), Tinospora cordifolia, Argemone mexicana (prickly poppy), and Eschscholzia californica 7. For pharmacological efficacy of berberine, Cicero and Baggioni reported that berberine has anti-inflammatory, antioxidant, neuroprotective, and cardiovascular protective effects, which might be used to manage chronic cardiometabolic disorders7. Kumar et al. reported that berberine has been used in Ayurvedic and Chinese medicine for its antimicrobial, antiprotozoal, antidiarrheal, and antitrachoma activity; it might ameliorate several disorders, including metabolic, neurological, and cardiological problems8. Lee et al. also reported that berberine improves memory impairment, alleviates memory-associated decreases in cholinergic immunoreactivity, restores brain-derived neurotrophic factor in the hippocampus of rats administered with scopolamine, and may improve cognitive function by stimulating cholinergic enzyme activity9. Actually, Lee et al. demonstrated that berberine decreases significantly the expression of IL-1β, TNF-α, and cyclooxygenase-2 mRNA in the hippocampus of rats administered with scopolamine9. Zhang et al. reported that berberine treatment significantly downregulates the production of pro-inflammatory cytokines and suppresses inflammatory responses in lipopolysaccharide (LPS)-stimulated macrophages through inhibition of NF-κB signaling1. Jiang et al. reported that berberine suppresses IL-1β secretion that was induced by the activation of the NLRP3 inflammasome in macrophages, which could be an important therapeutic target in atherosclerosis2.Berberine is a well-known quaternary ammonium salt that is usually found in the roots of such plants as 14.Immunity is important for human health. Inflammation is necessary in immune activity. Inflammatory mediators, such as NO, prostaglandins, and cytokines, play a significant role in immune reactions and inflammatory phenomena. Pro-inflammatory mediators play an important role in removing pathogens such as bacteria, fungus, and virus. Sometimes an unregulated infection might cause a syndrome of excessive cytokine production, i.e., “cytokine storm (hypercytokinemia)”Viral infection as well as bacterial infection might evoke massive production of pro-inflammatory mediators. In spite of modern developments in drugs and vaccines, there are still many intractable disorders caused by pathogens. Thus, it is reasonable to search for a material to modulate viral inflammation and virus-induced hypercytokinemia.21. For viral inflammation, we showed that flavonoids, such as quercetin, oroxylin A, and baicalein, exert anti-inflammatory effects on RAW 264.7 cells induced by poly I:C6.For reference, we previously reported how immuno-inflammatory activities of various natural products may affect macrophages22. In the current study, our data show that berberine alleviates the cytotoxic effects of poly I:C on RAW 264.7. Thus, berberine might be an inhibitor of apoptosis in macrophages stimulated by poly I:C.In these studies, poly I:C was used to bring about inflammatory reactions in mouse macrophages, which are a virus-induced cellular inflammation model. It is well known that dsRNA is produced in immune cell through viral replication and elicit cytotoxic responses23. This study shows that berberine significantly inhibited the production of NO, PGE2, Fas, GM-CSF, LIF, LIX, RANTES, and MIP-2 as well as calcium release and phosphorylation of p38 MAPK, ERK1/2, IkB-α, and STAT3 in poly I:C-stimulated RAW 264.7 cells. These results suggest that the anti-inflammatory effect of berberine in poly I:C-stimulated RAW 264.7 cells might involve the calcium signaling pathway, including activation of MAPK, NF-kB, and STAT3.In 2001, Alexopoulou et al. reported that mammalian toll-like receptor 3 could recognize dsRNA and gradually induce cytokine production via NF-kB and MAPK activation24. In 2006, Endo et al. reported that the start of LPS-induced inflammation cascade in lung might be triggered through ER stress-CHOP pathway including the activation p38 MAPK and NF-kB25. With CHOP-induced apoptosis in macrophages, Gotoh et al. reported in 2004 that ER stress-CHOP pathway is involved in NO-mediated apoptosis in LPS plus interferon-γ-stimulated macrophages26. Although NO is known to be necessary for various physiological processes, it means that NO might be a crucial key factor in ER stress-CHOP pathway in virus-stimulated macrophages. Moreover, Mory M. reported meaningfully in 2007 that NO depletes ER Ca2+, which consequently causes ER stress and leads apoptosis in macrophages27. An increase in cytosolic calcium is well known to activate CAMKII, which induces the apoptotic signaling cascade in macrophages including Fas induction and activation of STAT129.It is not yet fully explained how viral infection causes an ER stress with intracellular calcium release and CHOP expression. In relation to CHOP signaling pathway in stressed cells, Wang and Ron reported in 1996 that CHOP is phosphorylated and activated by p38 MAPK in stressed cells under conditions of stressChop, Stat1, Stat3, and Fas in poly I:C-stimulated RAW 264.7 cells. Hence berberine may be able to alleviate ER stress induced by dsRNA via the calcium-STAT pathway including activation of MAPK and NF-kB. But this study could not evaluate whether calcium signaling causes CAMKII activation in poly I:C-stimulated RAW 264.7 cells or not. These studies also could not verify exactly the effect of berberine on Type I interferons signaling in poly I:C-stimulated RAW 264.7 cells. It is meaningful that intracellular calcium is an important signaling molecule of ER stress and increases with Chop overexpression in poly I:C-stimulated RAW 264.7 cells.Our experimental results show that berberine inhibits the overexpression of Finally, our data mean that berberine exerts an anti-inflammatory effect related to its inhibition of NO, PGE2, Fas, GM-CSF, LIF, LIX, RANTES, and MIP-2 in poly I:C-stimulated RAW 264.7 cells via the ER stress-related calcium-CHOP/STAT pathway. The actual effect of berberine on viral inflammation deserves to be further studied.2 parameter assay kit from R&D Systems . Phospho-p38 MAPK Antibody (T180/Y182) (eBioscience 17-9078-42), Phospho-ERK1/2 Antibody  (eBioscience 12-9109-42), phospho-STAT3 Antibody (Tyr705) (eBioscience 12-9033-42), phospho-IkB-α Antibody  (eBioscience 12-9035-42), and CD95 Antibody (APO-1/Fas) (eBioscience 12-0951-83), Mouse IgG1 kappa Isotype Control (eBioscience 12-4714-81), and Mouse IgG2b kappa Isotype Control (eBioscience 12-4732-81) from Life Technologies Corporation . All other solutions for flow cytometric analysis from Thermo Fisher Scientific.We purchased DMEM, FBS, penicillin, streptomycin, PBS, and other tissue-culture reagents from Thermo Fisher Scientific . Poly I:C, berberine, baicalein, indomethacin, Griess reagent, and all other chemicals for cell culture from Sigma-Aldrich . Multiplex cytokine assay kits from Millipore ; Fluo-4 calcium assay kits from Molecular Probes ; Real-time RT-PCR kits from Bio-Rad ; Prostaglandin E2 humidified incubator. RAW 264.7 were cultured for 24 h, and cell viability was evaluated with the tetrazolium-based colorimetric assay (MTT) according to the previous study with a microplate reader (Bio-Rad)6.RAW 264.7 cells were obtained from the Korea Cell Line Bank . Cells were cultured in DMEM supplemented with 10% FBS containing 100 U/mL of penicillin and 100 µg/mL of streptomycin at 37 °C in a 5% CO6.After 24 h incubation, NO concentration in culture medium was measured by the Griess reaction as in previous studies with a microplate reader (Bio-Rad)2+ indicator used for the in-cell measurement of calcium signaling. After 18 h of treatment in 96-well plates, the intracellular calcium signaling from each well containing RAW 264.7 (1 × 105 cells/well) was identified using Fluo-4 NW Calcium Assay Kits (Thermo Fisher Scientific) following the protocol of our previous study6. Specifically, after incubating the cells with poly I:C and/or berberine for 18 h at 37 °C, we measured the intracellular calcium level using Fluo-4 assay with a spectrofluorometer  with excitation and emission filters of 485 nm and 535 nm, respectively.Fluo-4 AM is a well-known fluorescent Ca6. After 24 h incubation, the following cytokine productions were analyzed: GM-CSF; LIF; LIX; RANTES; MIP-2; MIP-1α.We did this assay with multiplex cytokine assay kits and a Bio-Plex 200 suspension array system (Bio-Rad) as described previously5 cells/well) were incubated in each well with or without berberine in poly I:C for 18 h. After 18 h incubation, total RNA of each well was isolated using NucleoSpin RNA kit . Then, cDNA of the RNA samples was produced using iScript cDNA Synthesis kit (Bio-Rad). The mRNA expression of Stat1, Stat3, Chop, and Fas were evaluated with real-time RT-PCR using an Experion Automatic Electrophoresis System (Bio-Rad) and Bio-Rad CFX 96 Real-Time PCR Detection System (Bio-Rad)6. Tbp was used for RNA normalization. The sequence of each primer set is showed in Table In 6-well plates, RAW 264.7 (3 × 105 cells/well) were incubated in each well with or without berberine in poly I:C for 24 h. After 24 h incubation, cells were harvested and washed with Flow Cytometry Staining Buffer (Thermo Fisher Scientific). Cells were stained with antibodies for phospho-p38 MAPK, phospho-ERK1/2, phospho-STAT3, phospho-IkB-α, and Fas according to the manufacture’s protocol as described previously21. Prior to antibody staining, cells were fixed and permeabilized using Fix Buffer I (Thermo Fisher Scientific) and Perm Buffer III (Thermo Fisher Scientific), respectively. Stained cells were analyzed on the Attune NxT flow cytometer (Thermo Fisher Scientific). Data were obtained from the mean fluorescent intensities of samples. Details for startup, proper calibration and operation of the Attune can be found in the Attune User Guide . A serial gating strategy used forward scatter versus side scatter plots and the target antibody expression plots. Unstained cells were used as the negative controls for gating. Mouse IgG1 kappa Isotype Control was used to confirm the specificity of phospho-p38 MAPK Antibody and Fas Antibody. Mouse IgG2b kappa Isotype Control was used to confirm the specificity of phospho-ERK1/2 Antibody, phospho-STAT3 Antibody, and phospho-IkB-α Antibody. For analysis of raw data, we used Attune NxT software (Thermo Fisher Scientific). Baicalein (25 µM) was used as a positive control.In 6-well plates, RAW 264.7 (3 × 104 cells/well) were incubated in each well with or without berberine in poly I:C for 24 h. After 24 h incubation, the production of PGE2 in each well containing RAW 264.7 cells was evaluated using Prostaglandin E2 parameter assay kit (R&D Systems) according to the manufacture’s protocol. The absorbance of the end product was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad)30.In 96-well plates, RAW 264.7 . In all cases, a


Drosophila the significance of the β-spectrin N-terminus, and explored its functional interaction with a CH2-localized L253P mutation that underlies the neurodegenerative disease spinocerebellar ataxia type 5 (SCA5). We report that pan-neuronal expression of an N-terminally truncated β-spectrin fails to rescue lethality resulting from a β-spectrin loss-of-function allele, indicating that the N-terminus is essential to β-spectrin function in vivo. Significantly, N-terminal truncation rescues neurotoxicity and defects in dendritic arborization caused by L253P. In vitro studies show that N-terminal truncation eliminates L253P-induced high-affinity actin binding, providing a mechanistic basis for rescue. These data suggest that N-terminal sequences may be useful therapeutic targets for small molecule modulation of the aberrant actin binding associated with SCA5 β-spectrin and spectrin-related disease proteins.Recent structural studies of β-III-spectrin and related cytoskeletal proteins revealed N-terminal sequences that directly bind actin. These sequences are variable in structure, and immediately precede a conserved actin-binding domain composed of tandem calponin homology domains (CH1 and CH2). Here we investigated in  Moreover, the mammalian β-III-spectrin isoform and the Drosophila homolog, β-spectrin, are required for morphogenesis and stability of the large, complex dendritic arbors extended by cerebellar Purkinje cells13 and dendritic arborization (da) sensory neurons14, respectively. Dominant mutations in β-III-spectrin cause the neurodegenerative disease spinocerebellar ataxia type 5 (SCA5)15, which is characterized by atrophy of the cerebellar cortex containing Purkinje cell dendrites16. A SCA5 missense mutation, L253P, localizes to the β-III-spectrin actin-binding domain (ABD) and induces a ~ 1000-fold increase in actin affinity17. In Drosophila, this mutation is neurotoxic, and reduces dendrite stability and arbor outgrowth in class IV da neurons14. The SCA5 L253P mutation highlights the importance of proper binding of β-spectrin to actin to support neuronal structure and function.β-spectrin, in a heterotetrameric complex with α-spectrin, binds actin filaments to form a spectrin-actin cytoskeleton that lines the cytoplasmic surface of the plasma membrane. Recent studies in neurons showed that the spectrin-actin cytoskeleton confers mechanical resiliency to axons18. Structural studies of numerous tandem-CH ABDs showed that in the absence of actin, CH1 and CH2 are bound to each other in a compact structural state22. Cryo-EM studies of the β-III-spectrin23, filamin A24 or utrophin25 ABD in complex with actin showed that actin binding is directly mediated by CH1. CH1 engagement with actin requires an ABD conformational “opening” in which CH2 is repositioned to expose an actin-binding site in CH1, and to prevent steric clash between CH2 and actin. The β-III-spectrin SCA5 L253P mutation, which localizes to CH2, shifts the ABD conformational equilibrium towards the open state23, explaining the increased actin affinity induced by L253P.β-spectrin proteins bind actin through an ABD composed of two calponin homology domains (CH1 and CH2) in tandem. The two CH domains are conserved evolutionarily in β-spectrins, and in spectrin-related proteins including α-actinin, filamin and utrophin25, while the filamin A N-terminus forms a helix-loop structure24. Truncation of the N-terminus of filamin A24 or utrophin27 decreases actin binding. Significantly, truncation of the N-terminal sequence preceding the β-III-spectrin CH domains abolishes actin binding23. The divergence of N-terminal sequences among spectrin-related proteins suggests the N-termini also confer unique functionality. Indeed, a recent study of the utrophin terminus suggests it directs binding of utrophin to specific actin structures in cells27. Further, studies of α-actinin28 and plectin29 suggest that N-terminal phosphorylation and calmodulin binding regulate actin binding. The in vivo significance of the β-III-spectrin N-terminus, in any species, has yet to be established.Intriguingly, the recent structural studies of tandem-CH ABDs bound to actin revealed N-terminal sequences, immediately preceding CH1, that also bind actin. Unlike the CH domains, these N-terminal sequences vary greatly in amino acid composition and secondary structure. In the actin-bound state, both the β-III-spectrin and utrophin N-termini take the form of an extended helixDrosophila transgenic lines, we examine the functional significance of the β-spectrin N-terminus in vivo. We further explore the functional interaction of the N-terminus with the SCA5, CH2-localized L253P mutation that induces high-affinity actin binding.Here, using new 18; the novel N-terminal α-helix identified by cryo-EM is composed of N-terminal residues S37–A52, and is contiguous with CH1 helix A that begins at residue D53, Fig. Drosophila β-spectrin  fused to the Drosophila β-spectrin coding sequence, with or without the N-terminal truncation  in the endogenous βspec gene. βspecem21 is a recessive, loss-of-function allele containing a nonsense mutation in the thirteenth spectrin-repeat domain31, resulting in low level expression of a truncated β-spectrin protein, Fig. βspecem21 allele causes lethality in males and homozygous females. In contrast females heterozygous for βspecem21 are healthy. Pan-neuronal expression of transgenic wild-type β-spectrin (βspecWT) rescued adult viability in ~ 25% of male progeny receiving the βspecem21 mutant X-chromosome, Table βspec∆N-WT) failed to rescue βspecem21 lethality. This indicates that the N-terminus is required for β-spectrin function in vivo. In addition, expression of N-terminally truncated β-spectrin (βspec∆N-WT) in heterozygous βspecem21 females reduced viability by ~ 30%. This indicates that the N-terminal truncation mutant is mildly neurotoxic. Moreover, raising Drosophila incubation temperature from 22 to 25 °C, to increase expression of the N-terminal truncation mutant, reduced survival by ~ 95%, Table βspec∆N-WT in the absence of βspecem21also caused lethality  failed to rescue lethality of βspecem21 males, and dominantly induced lethality in females heterozygous for βspecem21, Table βspecΔN-SCA5) did not induce lethality in female βspecem21 heterozygotes  did not reduce arborization, consistent with mild neurotoxicity observed in rescue assays at 22 °C.To determine how N-terminal truncation impacts SCA5-induced dendritic arborization defects, nts Fig. , consistDrosophila lines were generated containing UAS-transgenes, consisting of β-spectrin, with or without N-terminus, fused at the C-terminus to GFP. The transgenes encoding the β-spectrin-GFP fusion proteins were inserted into the attP154 genomic locus, as performed for the untagged β-spectrins. In rescue assays, the β-spectrin-GFP fusions performed similar to their untagged counterparts  of the full-length β-spectrin-GFP protein, Fig. 23.To evaluate the potential impact of N-terminal truncation on β-spectrin protein level, an additional set of ts Table , indicatd size 29 kDa of tβspecem21 heterozygotes expressing the toxic SCA5 mutant β-spectrin survive to adulthood, enabling evaluation of SCA5 mutant protein abundance in head extracts. For SCA5 β-spectrin with intact N-terminus, western blot analysis revealed that soluble SCA5 β-spectrin protein is ~ threefold more abundant than wild-type β-spectrin with intact N-terminus, Fig. We likewise evaluated the abundance of SCA5 β-spectrin protein, with and without N-terminus. At 18 °C, a fraction of female Further, the average level of soluble, N-terminally truncated SCA5 β-spectrin protein was 67% lower than wild-type β-spectrin with intact N-terminus, and 89% lower than SCA5 β-spectrin, Fig. We evaluated whether the SCA5 mutation or N-terminal truncation impacts the binding of β-spectrin to α-spectrin. Co-immunoprecipitation assays showed that neither the SCA5 mutation nor N-terminal truncation disrupts the co-association of the mutant β-spectrin proteins with α-spectrin in head extracts, Figs. 23, and suggested that this reflects the loss of an N-terminus that is structurally disordered when not bound to actin.To assess how N-terminal truncation impacts the folded state and stability of the SCA5 mutant β-spectrin, circular dichroism spectroscopy was performed. As previously reported, the β-III-spectrin L253P ABD with intact N-terminus showed a pronounced α-helical absorption profile consistent with a well-folded ABD, Fig. 17, the L253P ABD with intact N-terminus was entirely bound to F-actin at all actin concentrations tested (3–140 µM), Figs. 17, and threefold lower than the affinity of the wild-type ABD with intact N-terminus (74 µM). The binding affinity of the N-terminally truncated L253P ABD is higher than that of the N-terminally truncated wild-type ABD, which did not bind actin with measurable affinity.To determine whether N-terminal truncation impacts L253P-induced high-affinity actin binding, in vitro co-sedimentation assays were performed. Consistent with high-affinity binding previously reportedested 3–10 µM, Fig32. In this assay, HEK293T cells are transfected with a DNA construct consisting of the ABD fused to GFP, and a second construct consisting of the actin-binding peptide Lifeact fused to mCherry. FRET between GFP and mCherry results from the formation of a ternary complex consisting of the ABD-GFP and Lifeact-mCherry bound to an actin filament. Co-expression of the wild-type ABD and Lifeact-mCherry resulted in an average FRET efficiency of 3.9%, Fig. To confirm the in vitro co-sedimentation data, we employed a recently developed Förster resonance electron transfer (FRET) assay that detects the binding of the β-III-spectrin ABD to F-actin in live cellsDrosophila, we demonstrated that the N-terminus is essential for β-spectrin function in neurons. Moreover, we showed that the N-terminus is required for neurotoxicity and dendritic arborization defects induced by L253P. We further demonstrated that the N-terminus is required for L253P-induced high-affinity actin binding, which provides a mechanistic explanation for how N-terminal truncation rescues SCA5 neurotoxicity.The binding of β-spectrin to actin is required for the formation of the plasma membrane-associated spectrin cytoskeleton that is implicated in numerous neuronal functions. Here we investigated the in vivo functional significance of the β-III-spectrin N-terminus, which flanks the conserved tandem-CH ABD, and was recently discovered by cryo-EM, to bind actin. Using 34. Phosphorylation of the α-actinin N-terminus reduces actin binding28. Potentially β-spectrin phosphorylation modulates the function of the N-terminus to promote actin binding.The requirement of the N-terminus for L253P-induced high-affinity actin binding emphasizes the critical role of the N-terminus in the actin-binding mechanism. The N-terminus supports β-spectrin actin binding, at least in part, through its direct interaction with actin. The N-terminus is also destabilizing, Fig. 35. Cryo-EM of the filamin ABD-actin complex showed that in the “open” conformation, W142 makes hydrophobic contacts with actin24, avoiding solvent exposure. Actin binding may stabilize the L253P ABD by burying hydrophobic residues that would otherwise be solvent exposed. In contrast, the N-terminally truncated L253P ABD has very low affinity for actin, and thus remains destabilized and prone to misfolding and protein turnover.Here we showed that the SCA5 L253P mutation, in addition to causing high-affinity actin binding, also causes the mutant β-spectrin protein to accumulate in neurons, Fig. 32. The critical role of the N-terminus in actin binding suggests that small molecules that target the N-terminus may effectively reduce actin binding, similar to N-terminal truncation. In addition, it is possible that the large difference in stability of L253P β-III-spectrin versus wild-type may be leveraged to selectively target mutant β-III-spectrin. We suggest that the reduced protein level of N-terminally truncated SCA5 β-spectrin, but not N-terminally truncated wild-type β-spectrin, is due to enhanced protein turnover of a destabilized SCA5 β-spectrin that cannot bind actin. Thus, a small molecule that reduces actin binding may enhance turnover of the destabilized L253P β-III-spectrin, but not the more stable wild-type protein.A currently pursued therapeutic strategy for SCA5 is to identify small molecules that bind the L253P β-III-spectrin ABD and reduce its binding to actin477-Gal4/CyO, ppk-CD4-tdTom (10a)/TM6, Tb, and elav-Gal4 (C155). attP154 transgenic flies were kindly provided by Norbert Perrimon, Harvard Medical School, Boston. The βspecem21 line was obtained from Ronald Dubreuil, University of Illinois at Chicago, Chicago. We previously generated UAS-βspecWT and UAS-βspecSCA5 fly lines14.All stocks were maintained on standard food. The following stocks were obtained from the Bloomington Drosophila Stock Center: 14 were used as templates in PCR to delete the DNA sequence encoding the N-terminal 44 amino acids of Drosophila β-spectrin. For wild-type or SCA5 template, two PCRs were performed using the oligo set, AAAGGTACCGCCAAGTGAAGTTCATCC and CACACTCTCACGCTCCTCGGCCATGGCTGGGGATCTACGGTT, and the oligo set, AACAGATCCCCAGCCATGGCCGAGGAGCGTGAGAGTGTG and AAAGCTAGCTGCTCCAGTTTCTCCTGC. The resulting two PCR products contained overlapping sequences on either side of the intended deletion. To join the two PCR products, a third PCR was performed in which the two PCR products were used as template, together with the oligo AAAGGTACCGCCAAGTGAAGTTCATCC, which contains a KpnI site, and the oligo AAAGCTAGCTGCTCCAGTTTCTCCTGC, which contains a NheI site. The resulting PCR product contained the 5’ region of the wild-type or SCA5 β-spectrin cDNA, with the sequence encoding the N-terminal 44 amino acids deleted. The wild-type or SCA5 PCR product was then inserted KpnI-NheI into an intermediate DNA vector, pAc5.1b-FSPWT, containing the full-length Drosophila β-spectrin cDNA, digested with the same enzymes. The full-length cDNA encoding the N-terminally truncated wild-type or SCA5 β-spectrin was then subcloned from the pAc5.1b intermediate into pUASTattB using KpnI and XbaI enzymes. The final constructs, pUASTattB-β-spectrin ∆N-WT and pUASTattB-β-spectrin ∆N-SCA5 were sequence verified and inserted into the attP154 landing site using PhiC31 integrase-mediated transgenesis conducted by BestGene, Inc.The previously generated pUASTattB-β-spectrin wild-type and pUASTattB-β-spectrin SCA5 DNA constructsattP154 landing site at BestGene, Inc.A multi-step protocol was followed to fuse mEGFP to the 3′ end of the fly β-spectrin coding sequence. PCR were performed to amplify a 3′ region of the fly β-spectrin coding sequence, using pUASTattB-β-spectrin wild-type as template, together with forward primer, AAACGCCGGCGAGGGTCACGAAGG and the reverse primer, CTCCTCGCCCTTGCTCACCATCTTTTTCTTTAAAGTAAAAAACG. The reverse primer contains sequence complementary to the 3′ end of the β-spectrin coding sequence and sequence complementary to the 5′ end of mEGFP. A second PCR was performed to amplify mEGFP using the forward primer, ATGGTGAGCAAGGGCGAGGAG and the reverse primer, AAATCTAGACTCGTTCTTCTCTTGCTTATGGTTGCGTTACGGCTGTTACTTTACTTGTACAGCTCGTCCATGCC. The reverse primer includes a short β-spectrin 3’UTR sequence, attached to the 3′ end of the mEGFP coding sequence. The mEGFP DNA used as template in the PCR was synthesized by IDT DNA Technologies. A third PCR was performed to fuse the PCR products generated in the first two PCRs. In the third PCR, the PCR products from the first two PCRs were used as template, and combined with the forward primer, AAAGGTACCCGCCGGCGAGGGTCACGAAGG, containing KpnI and MreI restriction sites, and the reverse oligo AAATCTAGACTCGTTCTTCTCTTGCTTATGGTTGCGTTACGGCTGTTACTTTACTTGTACAGCTCGTCCATGCC, containing XbaI site. The resulting PCR product was a fusion of the 3′ coding sequence and mEGFP. The fusion DNA was inserted KpnI-XbaI into pcDNA3.1 digested with the same restriction enzymes, to generate the intermediate construct termed pcDNA3.1-fly-β-spec CT-GFP. The β-spectrin 3′ coding sequence with GFP fusion was subcloned from pcDNA3.1-fly-β-spec CT-GFP into pUASTattB-β-spectrin constructs using MreI and XbaI restriction enzymes. The final pUASTattB-β-spectrin-mEGFP constructs were sequence verified and transgenesis performed using the βspecem21, elav-Gal4, balanced over FM6, were crossed to male flies homozygous for UAS-βspec transgenes, or attP154 landing site, on the third chromosome. All classes of adult progeny were scored. Fly incubation temperature ranged from 18 to 25 °C, as indicated in the Tables containing rescue assay data.Female flies containing a recombinant X chromosome 477-Gal4; ppk-CD4-tdTom/TM6 transgene were crossed to UAS-βspec males at 22 °C. Eggs were collected from grape juice plates containing yeast paste every 4 h. Third-instar female larvae 120 h after egg laying (AEL) were imaged using HC PL APO 20x/0.80 NA dry objective and and a Leica DMi8 inverted microscope equipped with X-Light V2 spinning-disc (CrestOptics), LDI laser unit (89 North), and Photometrics Prime 95B CMOS camera. Z-stack images of class IV da neurons were obtained from A3 and A4 abdominal segments (1–3 neurons per larva) and processed to generate maximum intensity projections using Metamorph software. Reconstructions of the arbors were generated using Photoshop 21.0.1. Sholl analysis, and total branch length and branch point quantitation were performed as previously described using ImageJ software36. Statistical analyses on total branch length and number of branch points were performed in Prism 8 (Graphpad) using unpaired two-tailed t test .In Fig. UAS-βspec-GFP transgenes or attP154 landing site were crossed to females containing βspecem21, elav-Gal4/FM6. Flies were incubated on standard food at 18 °C. Adult female progeny containing βspecem21, elav-Gal4 and UAS- βspec-GFP transgenes were collected and stored at − 80 °C. To separate fly heads from bodies, a microtube containing flies was frozen in liquid nitrogen and then vigorously shaken. Heads were collected and homogenized using a G-tube with pestle  in RIPA buffer , 1% IGEPAL CA-630, and 0.7% DOC) at a ratio of 2 µL buffer per head. The supernatants containing soluble proteins were mixed with 4X Laemmli sample buffer (Bio-Rad) after centrifugation at 17,000 RCF and 4 °C for 15 min. The pellets (insoluble fraction) were resuspended in 1X sample buffer. SDS-PAGE was performed on the soluble and insoluble protein samples using 12% SDS-acrylamide gels. Proteins were transferred from gels to Immobilon-FL membranes (Millipore). Membranes were blocked with 1% casein  in 1 × PBS-L  for 1 h at room temperature. Then the membranes were probed overnight at 4 °C with the following primary antibodies diluted in 1 × PBS-L with 1% casein and 0.1% Tween 20: 1:2000 α-actin JLA20 , 1:2000 α-GFP (Millipore) or 1:1000 Drosophila β-spectrin antibody. Primary antibody solution was removed and membranes washed with 1 × PBS plus 0.1% Tween 20. The membranes were probed for 1 h with the following secondary antibodies: α-mouse IRDye 800CW (LI-COR Biosciences), α-rabbit IRDye 680RD (LI-COR Biosciences), or α-guinea pig IRDye 680RD (LI-COR Biosciences), diluted to 1:5000 in 1xPBS-L with 1% Casein, 0.1% Tween 20, and 0.01% SDS. Secondary antibodies were removed and membrane washed with 1 × PBS plus 0.1% Tween 20. Membranes were imaged on a Sapphire scanner (Azure) using 680 nm and 800 nm channels. Protein band fluorescence intensities were quantified in ImageStudio lite (LI-COR Biosciences) by drawing rectangular boxes of uniform size around each protein band. Local fluorescence background signal was subtracted using the median pixel intensity measured under the upper and lower bounds of each measurement box.Males homozygous for Drosophila head extracts were prepared as described above. After, homogenization and clarification by centrifugation, a Bradford assay was performed to determine lysate protein concentrations. A sample of each lysate (80–100 µg) was collected and mixed with 2 × Laemmli sample buffer. Remaining lysate supernatants were clarified by inversion with Protein A agarose resin for 1 h at 4 °C. Then ~ 500 µg total lysate protein was incubated with either 10 µg anti-Drosophila α-spectrin 3A9  or 10 µg normal mouse IgG (Millipore) antibodies for 1 h at 4 °C, with inversion. Protein A agarose beads equilibrated with RIPA buffer was then added to lysate/antibody mixtures and incubated with inversion overnight at 4 °C. Then, Protein A agarose beads were pelleted at 760×g for 1 min and washed 3 × with RIPA buffer. The protein samples were eluted by boiling the beads with 1X Laemmli sample buffer for 5 min at 85 °C.17 using the following forward primers for intact N-terminus AAACACCTGCAAAAAGGTATGAGCAGCACGCTGTCACCC or truncated N-terminus AAACACCTGCAAAAAGGTGCAGATGAACGAGAAGCTGTGC, and the reverse primer AAATCTAGACTACTTCATCTTGGAGAAGTAATGGTAGTAAG. PCR products were digested with AarI and XbaI restriction enzymes and ligated into the compatible ends in pE-SUMOpro (LifeSensors) generated by BsaI digestion. The final constructs, pE-SUMO-ABD L253P and pE-SUMO-A52-ABD L253P, together with the previously generated pE-SUMO-ABD WT and pE-SUMO-A52-ABD WT constructs23, were transformed into Rosetta 2 (DE3) E. coli (Novagen). Bacteria were gown in 1 L of LB broth with ampicillin (100 µg/mL) and chloramphenicol (34 µg/mL) and SUMO-ABD protein expression induced with 0.5 mM IPTG, for 6 h, at 300 rpm and room temperature. Bacteria were pelleted at 4000 rpm and 4 °C for 30 min, and pellets stored at − 20 °C until further use. Protein extraction and purification proceeded as described previously23, except that a final gel filtration chromatography step was not performed due high purity of ABD protein. Buffer exchange was performed by dialysis in 10 mM Tris, pH 7.5, 150 mM NaCl, 2 mM, MgCl2, and 1 mM DTT using Slide-A-Lyzer, 10,000 MWCO cassettes (Thermo Scientific).The coding sequences for L253P β-III-spectrin ABD with or without N-terminus were PCR amplified from pET-30a-ABD L253P2, and 1 mM DTT. Circular dichroism absorption spectra and thermal denaturation data were collected in a Jasco J-810 spectropolarimeter with Peltier temperature controller, as described previously23. Raw absorption data was converted to mean residue ellipticity using an equation described previously23. For thermal denaturation absorption data measured at 222 nm, melting temperatures were calculated in Prism 8 (Graphpad) using nonlinear regression to fit a two-state transition equation previously described23.Purified ABD proteins were diluted to 200 ng/µL in buffer containing 10 mM Tris, pH 7.5, 150 mM NaCl, 2 mM, MgCl2. Homogenized actin was clarified by centrifugation at 70,000 RPM and 4 °C for 10 min in a TLA-100.3 rotor. Actin was polymerized to F-actin by addition of 2 mM MgCl2 for 30 min at room temperature. F-actin was pelleted at 80,000 RPM for 30 min at 4 °C in a TLA-100.3 rotor. The F-actin was resuspended and homogenized in F-buffer containing 10 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM ATP, 2 mM MgCl2, 1 mM DTT. Immediately before binding assays, ABD proteins were clarified by centrifugation at 40,000 RPM for 30 min at 4 °C in a TLA-100.3 rotor. ABD and F-actin concentrations were determined by Bradford assay. Binding reactions were performed by combining 2 µM ABD with varying concentrations for F-actin (3–160 µM), and incubating reactions for 30 min at room temperature. To pellet F-actin and actin-bound ABD, binding reactions were centrifuged at 50,000 RPM, for 30 min at 25 °C, in a TLA-100 rotor (Beckman). Supernatants containing unbound ABD protein were sampled and mixed with 1X Laemmli sample buffer. In Fig. 17.Actin was purified from rabbit muscle acetone powder acquired from Pel-Freez Biologicals. To extract actin, acetone powder was incubated in pre-chilled water (1 g / 20 mL) for 30 min followed by vacuum-filtration using Whatman #4 filter paper. One Complete protease inhibitor cocktail, without EDTA, mini tablet (Roche) was added to the filtrate. Actin was then polymerized to F-actin by addition of 30 mM KCl for 1 h at room temperature. F-actin was pelleted by centrifugation at 80,000 RPM at 4 °C, for 30 min, in a TLA-100.3 rotor (Beckman). Supernatants were decanted and F-actin depolymerized by resuspension and homogenization in G-buffer containing 5 mM Tris, pH 7.5, 0.5 mM ATP, and 0.2 mM CaCl2, 1 mM DTT. BSA protein was prepared at concentration of 2 µM, also in F-buffer. Samples were mixed with 2X Native Sample Buffer (Bio-Rad). Native-PAGE was performed using a 12% acrylamide gel and ice-cold Tris–glycine buffer (Bio-Rad). Gels were stained using Coomassie Brilliant Blue R-250 solution. The destained gel was imaged using the 785 nm channel of an Azure 600 imager.ABD and BSA proteins were clarified by centrifugation at 40,000 RPM for 30 min at 4 °C in a TLA-100.3 rotor. ABD and BSA concentrations were determined by Bradford assay. Varying concentrations of ABD proteins (2–8 µM) were prepared in F-buffer containing 10 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM ATP, 2 mM MgCl32. HEK293T cells were acquired from the American Tissue Culture Collection (ATTC) and were transiently transfected using Lipofectamine 3000 (ThermoFisher Scientific) with either pcDNA3.1-β-III-spectrin ABD-mEGFP donor construct (D), or co-transfected with pcDNA3.1-β-III-spectrin ABD-mEGFP and pcDNA3.1-Lifeact-mCherry donor and acceptor constructs (DA), in 6-well microplates. Cells were harvested 24 h after transfection by dissociation with TrypLE (Gibco), followed by TrypLE inactivation in 1 × DMEM (4.5 g/L D-glucose and 110 mg/L sodium pyruvate) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco), and then washing in 1 × DPBS (Gibco). Fluorescence lifetime measurements were performed using time-correlated single photon counting method in a FS5 spectrofluorometer equipped with an EPL-472 pulse laser with 5 MHz frequency (Edinburgh Instruments). mEGFP emission signals were detected at 510 nm until the fluorescence decay reached 1000 photon counts within a 50 ns time window. The lifetime (τ) of mEGFP in D and DA samples was obtained by fitting the fluorescence decay data using Fluoracle software (Edinburgh Instruments) reconvolution analysis with instrument response function correction. The FRET efficiency was calculated using the equation:Mammalian cell DNA constructs were generated to express ABD proteins fused at the C-terminus to mEGFP, to be used as FRET donors. For these constructs, the mEGFP coding sequence was PCR amplified using the forward primer CTTGGTACCACCATGGTGAGC, containing a KpnI site, and the reverse primer AAATCTAGACTACTTGTACAGCTCGTCCATGCC, containing a XbaI site, followed by digestion and ligation into pcDNA3.1, resulting in the intermediate construct pcDNA3.1-mEGFP. For PCR amplification of the ABD coding sequence, the forward primer AAAAAGCTTACCACCATGAGCAGCACGCTGTCACCC, or AAAAAGCTTACCACCATGGCAGATGAACGAGAAGCTGTGC, for full-length or N-terminally truncated ABD, respectively, was used with the reverse primer AAAGGTACCCTTCATCTTGGAGAAGTAATGG. The amplified ABD sequences were digested with HindIII and KpnI, and ligated 5′ of mEGFP in pcDNA3.1-mEGFP. The final FRET donor constructs pcDNA3.1-β-III-spectrin ABD-mEGFP with or without the N-terminal truncation were sequence verified. The pcDNA3.1-Lifeact-mCherry FRET acceptor construct was previously describedFor Fig. Supplementary Information.