Pseudomonas syringae, Pseudomonas aeruginosa, Xanthomonas oryzae and Ralstonia solanacearum) and a clinical isolate (Pseudomonas aeruginosa UCBPP-PA14) under nutrient-limiting conditions. We proposed a luminescence reporter system to quantitatively determine the translational elongation rates (ERs) of T3SS regulators, effectors and structural protein under different nutrient-limiting conditions and culture durations.Sufficient nutrition contributes to rapid translational elongation and protein synthesis in eukaryotic cells and prokaryotic bacteria. Fast synthesis and accumulation of type III secretion system (T3SS) proteins conduce to the invasion of pathogenic bacteria into the host cells. However, the translational elongation patterns of T3SS proteins in pathogenic bacteria under T3SS-inducing conditions remain unclear. Here, we report a mechanism of translational elongation of T3SS regulators, effectors and structural protein in four model pathogenic bacteria .Rapid translational ER and synthesis of T3SS protein need adequate tRNAs and EFs in nutrient-limiting conditions. Numeric presentation of T3SS translation visually indicates the invasion of bacteria and provides new insights into T3SS expression that can be applied to other pathogenic bacteria.The online version contains supplementary material available at 10.1186/s13578-022-00884-6. Bacterial growth primarily depends on protein synthesis catalyzed by the ribosome. Protein accounts for half of the bacterial biomass, and its synthesis requires more than 60% of the total cellular energy , 2. BactPseudomonas syringae, Pseudomonas aeruginosa, Xanthomonas oryzae and Ralstonia solanacearum, the highly conserved T3SS is the key factor allowing the establishment of infection. The T3SS needle-like machinery delivers effector proteins into host cells to suppress the host immune system [The activity of the bacterial type III secretion system (T3SS) is regulated by environmental nutrition conditions or the host environment and enables bacterial pathogens to invade host cells , 20. In e system , 22. Bace system –25, while system , 27. In e system –30.P. aeruginosa, the T3SS genes are induced by contact with host cells or by low-calcium conditions, which can be achieved by adding 5 mM ethylene glycol tetraacetic acid (EGTA) to LB [popN-pcr1-pcr2-pcr3-pcr4-pcrD-pcrR-pcrG-pcrV-pcrH-popB-popD. PopN contributes to detecting the intracellular calcium levels [P. aeruginosa [Many T3SS regulators have been identified in recent decades –34. In tA) to LB , 36. TheA) to LB . ExsA trA) to LB , 38. Popm levels . PopN inruginosa , 40.hrc) and pathogenicity (hrp) genes, which are divided into two groups. Group I includes the hrp clusters of P. syringae, while group II contains those of X. oryzae and R. solanacearum [hrp box in the T3SS gene promoter region [hrpL transcription is activated through the regulation of another alternative sigma factor, RpoN [hrpL transcription is also autoregulated through the bi-directional promoter region between hrpL and hrpJ [hrpL promoter and activates hrpL expression [hrpRS gene operon is positively regulated by HrpA, a component of the T3SS pilus, and the extra-cytoplasmic function sigma factor AlgU [P. syringae pv. tomato DC3000 [hopG1 gene encodes a T3SS effector protein that localizes to the mitochondria. HopG1 promotes the growth of pathogens and inhibits the plant defense responses [P. syringae pv. tabaci, HopX1 facilitates the susceptibility through directly degrading jasmonate pathway repressors [In plant pathogens, T3SS is encoded by the clustered hypersensitive response , P. aeruginosa (PA), X. oryzae (XO) and R. solanacearum (RS). Here, we constructed two kinds of reporters. One included the corresponding promoter and a promoter-less luciferase (lux) in the plasmid, which was the transcriptional fusion reporter to calculate the transcription time (Tinitiation). The other reporter included the corresponding promoter, the coding sequence (CDS) and a promoter-less luciferase (lux) in the plasmid, which was the reporter for both transcription and translation to calculate the time for both processes (Ttest). The translational elongation time (Tfirst) of target protein was calculated by Ttest minus Tinitiation. The translational ERs of the tested proteins equaled protein length (L) divided by Tfirst [The translational ER is an accurate indicator for evaluating protein synthesis . Elongatlow rate . Howevery Tfirst , 75.RS for RS) and T3SS-suppressing conditions , we constructed eight transcriptional and translational fusion reporters containing the full-length CDS of the following T3SS genes: hrpLPS, hopG1PS, hrpRPS, hopX1PS, hrpA2PS, popNPA, hpa1XO and hrpGRS. As shown in Fig. PS and PopNPA increased rapidly within the first 3 h in T3SS-inducing conditions  were induced to a high level in the nutrient-rich media , a highly virulent and infectious strain [PA, PopNPA14 was significantly induced by threefold in LB with 5 mM EGTA within the first 3 h, and gradually reduced within the following 7 h  and higher  than the normal T3SS-inducing medium (1× T3SS-inducing medium). The results showed that the T3SS translational ERs of PS, XO and RS in 0.5× MM or 0.5× XOM2 were higher  than those in 1× MM or 1× XOM2  was higher than that in the 2× rich medium and nutrient-limiting medium (less than 1.5 aa/s) , and all the AdhBPS ERs in MM were low, with no significant differences among them (0.16–0.39 aa/s)  were also higher than those found in medium supplemented with EGTA (0.4–3.5 aa/s)  were higher than those at 50 and 60 min , and were less than 1.0 aa/s in LB supplemented with EGTA  had no significant differences with those in 0.5× M210 and 0.125× M210 (~ 0.8 aa/s). The ERs at 10, 30, 40 and 50 min (~ 0.7–0.8 aa/s) showed no differences with that at 0 min (~ 0.5 aa/s) apart from those at 20 and 60 min (~ 0.9–1.0 aa/s)  were much lower than that in 1× B (~ 13.4 aa/s). The translational ERs for 10–60 min (~ 1.3–2.7 aa/s) were much lower than that at 0 min (~ 13.4 aa/s)  from 0 to 16 min after induction and then kept at a low level, showing a time-course behaviour Fig. E. The trhrpLPS and hopG1PS mRNA transcription required ~ 1080 s and ~ 900 s, which were shorter than the initial first full-length protein syntheses    , leading to their translation at ~ 47.8 min and ~ 4.9 min  than the respective EFs (by ~ 60%) in nutrient-limiting conditions  1448A, P. aeruginosa (PA) PAO1, P. aeruginosa UCBPP-PA14 (PA14) PA14, X. oryzae (XO) PXO99A and R. solanacearum (RS) OE 1-1. The tested genes were hrpLPS, hopG1PS, hrpRPS, hopX1PS, hrpA2PS, adhBPS, popNPA, alpAPA, popNPA14, mexAPA14, hpa1XO, gumBXO, hrpGRS and sucCRS. The pMS402 plasmid was used [lux). The bacterial strains, plasmid and primers are listed in Table S1 in the Additional file lux) in the plasmid, which was the transcriptional fusion reporter. The other reporter concluded the corresponding promoter, the coding sequence (CDS) and a promoter-less luciferase (lux) in the plasmid, which was the transcriptional and translational fusion reporter.The bacterial strains used in this study were wild-type was used , which e600) of ~ 0.6. PA PAO1 and PA14 were cultured in LB at 37 °C. For PS, XO and RS, the pre-culture was centrifuged, washed three times, and resuspended in distilled water. For the induction curve, the lux activity of the final experimental culture at OD600 ~ 0.1 in the T3SS-inducing condition was immediately measured. The nutrient-limiting media were MM for PS 2SO4, 1.7 mM MgCl2, 1.7 mM NaCl, and 10 mM fructose, pH 5.7) [2PO4, 40 μM MnSO4, 240 μM Fe(III) EDTA and 5 mM MgCl2, pH 6.5) [RS for RS [2 were added to induce T3SS gene expression, and various doses of EGTA and MgCl2 were used to achieve a range of calcium stress conditions. Related parameters including the translational ER and the mRNA, tRNA and EF levels were measured at specific time points. The kanamycin concentrations were 100 μg/mL for PS and 50 μg/mL for XO and RS. The trimethoprim concentration was 50 μg/mL for PA and PA14.PS 1448A, XO PXO99A and RS OE 1-1 were grown in the nutrient-rich media King’B (KB) , M210 9 and B 9, respect pH 5.7) , XOM2 fo pH 6.5)  and MMRSS for RS , respectlux) in the plasmid, which was the transcriptional reporter to calculate the transcription time (Tinitiation). The other reporter concluded the corresponding promoter, the CDS and a promoter-less luciferase (lux) in the plasmid, which was the reporter for both transcription and translation to calculate the time for both processes (Ttest). The synthesis cost of the promoters was assessed as the initiation time, and the associated calibrations are described in Additional file 2 (for PA and PA14), the lux activity was measured using a microplate reader (600 nm excitation filter) at 2.5-min intervals. The induction curve of the tested proteins was constructed by plotting the lux activity against the induction time and analysed with a Schleif plot. We considered E(0) as the basal lux activity of the reporter and E(t) as the lux activity at the specific time point of induction. Ttest was regarded as the intact cost time, and Tinitiation was regarded as the initiation cost time. The synthesis time of the first round of the tested proteins, Tfirst, could thus be calculated as:The measurement and calculation of the translational ER were based on a luminescence reporter system and a LacZ induction assay, respectively , 75. Theith time ; therefoTherefore, the translational ER can be calculated as:XO and HrpGRS were showed in Additional file The mentclass2pt{minim600 ~ 0.6 and then transferred into the investigated media. Immediately after induction, 500 μL of culture was removed at 1-min intervals and added to 500 μL of pre-cooled stop solution containing 60% ethanol, 2% phenol and 10 mM EDTA. The total RNA purification of PS, PA, PA14 and XO was performed with a RNeasy mini kit (Qiagen); the RS total RNA was isolated using TRIzol reagent (Invitrogen). The RNA concentrations were measured using a NanoDrop 2000 spectrophotometer (ThermoFisher). cDNA synthesis was performed using HiScript III RT SuperMix . qRT-PCR was performed with a SuperReal Premix Plus (SYBR Green) kit (Tiangen Biotech) according to the manufacturer’s instructions. The relative mRNA abundance at each time point was 2Ct(0)−Ct(t), where Ct (0) indicates the Ct value of the sample taken immediately before transfer to nutrient-limiting conditions, and Ct (t) indicates the Ct value at each time point thereafter. The relative mRNA synthesis level was plotted against time to obtain the transcriptional kinetics curve.The mRNA, tRNA and EF abundances were measured by quantitative reverse transcription (qRT-PCR). The qRT-PCR primers used are shown Table S1 in the Additional file gyrB of XO and serC of RS were used as internal references. Relative expression levels of tRNAs and EFs at various time points were calculated by the 2−(ΔΔCt) method.To measurement the relative tRNA and EF levels, 500 μL of culture was removed at 8-min (PS) or 10-min  intervals and centrifuged at 12,000 rpm for 1 min to harvest the bacteria. The RNA purification and reverse transcription processes were performed as previously mentioned, except the total RNA was incubated at 80 °C for 15 min before cDNA synthesis to remove the secondary structure of tRNA. The corresponding 16S rRNAs of PS, PA and PA14, Additional file 1: Figure S1. Induction curves of PopNPA14 and non-T3SS proteins  induced in 1× T3SS-inducing conditions and 1× T3SS-repressing conditions. (A) The red curve showed the delayed expression levels of adhBPS-pro-CDS-lux reporter in 1× MM compared with that in 1× KB. (B) The red curve showed the slightly fast expression levels of alpAPA-pro-CDS-lux reporter in LB + 5 mM EGTA compared with that in LB. (C) The red curve showed the delayed expression levels of gumBXO-pro-CDS-lux reporter in 1× XOM2 compared with that in 1× M210. (D) The red curve showed the delayed expression levels of sucCRS-pro-CDS-lux reporter in 1× MMRS compared with that in 1× B. (E) The red curve showed the slightly fast expression levels of popNPA14-pro-CDS-lux reporter in LB + 5 mM EGTA compared with that in LB. (F) The red curve showed the slightly fast expression levels of mexAPA14-pro-CDS-lux reporter in LB + 5 mM EGTA compared with that in LB. (G) The expression of HrpGRS in MMRS at 50 and 60 min were induced rapidly so that the synthesis time of HrpGRS was not calculated. Figure S2. Expression levels of T3SS regulators, effectors and structural protein of P. syringae, X. oryzae and R. solanacearum in 0.5× T3SS-inducing conditions were less stable than that in 1× T3SS-inducing conditions. The red curve and green curve denoted the expression levels in 1× and 0.5× T3SS-inducing conditions, respectively. (A) The expression levels of hrpLPS-pro-CDS-lux reporter were extremely low in 0.5× MM compared with those in 1× MM.  The expression levels of hopG1PS, hrpRPS, hopX1PS and hrpGRS-pro-CDS-lux reporters in 0.5× T3SS-inducing conditions rapidly increased, but declined to a low level immediately compared with those in 1× T3SS-inducing conditions. (E) The expression levels of hpa1XO-pro-CDS-lux reporter in 0.5× XOM2 were slightly lower than those in 1× XOM2. Figure S3. Translational elongation rate of PopNPA14 and non-T3SS proteins  in multiple nutrition conditions and at various time. (A-B) Translational ERs of PopNPA14 under different EGTA concentrations and at various culture time (0–60 min). (C) The translational ERs of MexAPA14 in LB with EGTA (~ 1.0- ~ 2.2 aa/s) were much lower than that in LB (~ 5.2 aa/s) with no significant difference. (D-E) The translational ERs of MexAPA14 in both LB and LB with 5 mM EGTA during various culture times kept at a low level . (F) The translational ER of AdhBPS in 1× MM (~ 8 aa/s) was more than fourfold than those in richer or poorer nutrition conditions (less than 2 aa/s). (G) In 1× KB, the translational ER of AdhBPS at the beginning of culture (~ 8 aa/s) had significant difference than those at various culture time (less than 2 aa/s). (H) In 1× MM, the translational ERs of AdhBPS at 0–4 h of culture were much lower than that in 1× KB, which had no difference. (I) The translational ER of AlpAPA in LB (~ 13 aa/s) was more than threefold than those in LB + EGTA (less than 5 aa/s). (J) In LB, the translational ERs of AlpAPA at 0–40 min of culture (10–15 aa/s) had no difference. The ER decreased rapidly at 50–60 min (less than 5 aa/s). (K) In LB + 5 mM EGTA, the translational ERs of AlpAPA were much lower than those in LB. (L) The translational ER of GumBXO in 1× M210 (~ 0.5 aa/s) had no significant differences with those in 0.5× M210 and 0.125× M210 (~ 0.8 aa/s). (M) The translational ERs of GumBXO at 10, 30, 40 and 50 min (~ 0.7–0.8 aa/s) showed no differences with that at 0 min (~ 0.5 aa/s) apart from those at 20 and 60 min (~ 0.9–1.0 aa/s). (N) The ERs of SucCRS in 0.5× , 0.25× and 0.125× B (~ 0.9–1.4 aa/s) were much lower than that in 1× B (~ 13.4 aa/s). (O) The translational ERs of SucCRS for 10–60 min (~ 1.3–2.7 aa/s) were much lower than that at 0 min (~ 13.4 aa/s). Figure S4. Induction assay of full-length PopNPA14 and non-T3SS proteins  mRNAs in 1× T3SS-inducing conditions. (A) The mRNA synthesis level of popNPA14 was rapidly induced in LB with 5 mM EGTA. Ttest of PopNPA14 was ~ 2191 s in LB with 5 mM EGTA. (B) The mRNA synthesis level of adhBPS rapidly decreased in 1× MM and maintained a low level within 26–420 min. Ttest of AdhBPS was ~ 343.7 min in 1× MM. (C) The mRNA synthesis level of alpAPA increased after adding EGTA and dropped to the starting level. Ttest of AlpAPA was ~ 47 s in LB + 5 mM EGTA. (D) The mRNA synthesis level of mexAPA14 increased after adding EGTA and dropped to the starting level. Ttest of MexAPA14 was ~ 5 min in LB + 5 mM EGTA. Figure S5. Relative expression levels of tRNAs and EFs of PA14 in LB with 5 mM EGTA. (A-C) tRNAs of PA14 were detected at 10, 20, 30, 40, 50, 60 min. The data at 0 min for each individual tRNA was set as 1. (D) The relative EFs levels of PA14 were detected by qRT-PCR. The EFs levels were shown together with the translational ER of PopNPA14 in LB with 5 mM EGTA. The translational ERs at specific times were shown as green hollow symbols. The standard deviations were shown but were very small in the plot.  *p < 0.05; **p < 0.01; and ***p < 0.001. Results were indicated in mean ± SD. All experiments were repeated at least three times. Figure S6. The expression levels of T3SS genes during 0–6-h inducing culture. (A–E) The expression levels of hrpLPS, popNPA, hpa1XO, hrpGRS and popNPA14 in 1× T3SS-inducing conditions for 0–6 h. Figure S7. The Schleif plot of T3SS regulators, effectors and structural protein of P. syringae, P. aeruginosa, X. oryzae and R. solanacearum in 1× T3SS-inducing conditions. The Schleif plot was used to detect the translational time of the first newly synthesized protein in 1× T3SS-inducing conditions. The square root of newly synthesized protein (E(t) denoted the expression levels of the tested proteins at specific times in 1× T3SS-inducing conditions, and E(0) denoted the basal expression levels of the culture. The green line and red line described the linear line of transcriptional fusion reporters and reporters containing both transcription and translation, respectively. The x-intercepts of the green line and red line denoted the time for the initial cost (Tinitiation) and the transcription and translation of the tested proteins (Ttest), respectively. Therefore, the difference of the two intercepts corresponded to the time to translate the full-length protein molecule (Tfirst). The initial cost included the sense of cells to nutrient conditions, RNA polymerase transcriptional initiation and ribosome translational initiation (Ref. 4 of the main text). The initiation time of various proteins were different . Figure S8.Ttests of Hpa1XO and HrpGRS in 1× T3SS-repressing conditions were much longer than those in 1× T3SS-inducing conditions. The red line and blue line denoted the time containing both transcription and translation (Ttest) in 1× T3SS-inducing conditions and 1× T3SS-repressing conditions, respectively. (A) Ttests of hpa1XO-pro-CDS-lux reporter in 1× XOM2 and 1× M210 were ~ 3.352 min and ~ 300.4 min. (B) Ttests of hrpGRS-pro-CDS-lux reporter in 1× MMRS and 1× B medium were ~ 42.35 s and ~ 181.2 s. Table S1. Bacterial strains, plasmid, and primers used in this study.


The generation of reactive oxygen species (ROS) can affect cationic liposome-mediated transfection. In this study, we focused on a specific class of antioxidants, flavonoids, to investigate the transfection efficiency using cationic liposome/plasmid DNA complexes (lipoplexes) in 2D and 3D cultures of Colon26 and HepG2 cells, respectively. All tested flavonoids enhanced the transfection efficiency in 2D Colon26 and HepG2 cells. Among the tested flavonoids, 25 µM quercetin showed the highest promotion effect of 8.4- and 7.6-folds in 2D Colon26 and HepG2 cells, respectively. Transfection was also performed in 3D cultures of Colon26 and HepG2 cells using lipoplexes with quercetin. Quercetin (12.5 µM) showed the highest transfection efficiency at all transfection timings in 3D Colon26 and HepG2 cells with increased cell viability. Flow cytometry revealed that quercetin treatment reduced the population of gene expression-negative cells with high ROS levels and increased the number of gene expression-positive cells with low ROS levels in HepG2 cells. Information from this study can be valuable to develop strategies to promote transfection efficiency and attenuate cytotoxicity using lipoplexes. N-acetyl cysteine, and water-insoluble antioxidants vitamins A and E. Here, flavonoids, ubiquitous in food and plant sources, play an important role as antioxidants, gaining more advantages in terms of economic benefits and sustainable use. Flavonoids decrease ROS levels caused by H2O2 [Gene therapy is an option for cancer treatment. The development of a safe and effective gene delivery system is important for successful gene therapy. Compared to viral vectors, non-viral vectors possess lower immunogenicity, indicating their potential for widespread application worldwide . Among n by H2O2 ,7,8. In  by H2O2 ,10. To d by H2O2 ,12. Nano by H2O2 ,14. We hEpigallocatechin, myricetin, fisetin, quercetin, kaempferol, galangin, naringenin, and epigallocatechin gallate were purchased from the Tokyo Chemical Industry Co. . Lipofectamine 3000, CellROX Orange, and CellROX Deep Red were purchased from Thermo Fisher Scientific , and the autophagy detection reagent, DALGreen, was purchased from Dojindo Laboratories .Gaussia luciferase, ptdTomato-C1 encoding the red fluorescent protein tdTomato, and pTagBFP-N encoding the blue fluorescent protein TagBFP were purchased from Lux Biotechnology Ltd. , Clontech , and Evrogen Joint Stock Company , respectively. Amplification of plasmid DNA was performed using the Escherichia coli strain, DH5α. Plasmid DNA was purified using the EndoFree Plasmid Giga Kit . Cy5-labeled plasmid DNA was prepared using the Label IT Nucleic Acid Labeling Kit .pcDNA3/GL encoding secretable 2 at 37 °C. HepG2 cells were grown under standard conditions in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% FBS, penicillin G (100 units/mL), and streptomycin (100 µg/mL) in a humidified atmosphere with 5% CO2 at 37 °C.Colon26 and HepG2 cells were obtained from RIKEN . Colon26 cells were grown under standard conditions in Roswell Park Memorial Institute 1640 medium  supplemented with 10% fetal bovine serum (FBS), penicillin G (100 units/mL), and streptomycin (100 µg/mL) in a humidified atmosphere with 5% COFor epigallocatechin, treatment with less than 100 µM epigallocatechin could improve the apoptosis of PC12 cells, which were induced by 6-hydroxydopamine exposure ; incubatAccording to the above references and results of our preliminary experiments, we finally decided on flavonoids concentrations as epigallocatechin (1.56–50 µM), myricetin (0.31–10 µM), fisetin (1.56–50 µM), quercetin (6.25–100 µM), kaempferol (3.13–100 µM), galangin (6.25–100 µM), naringenin (5–80 µM), and epigallocatechin gallate (6.25–100 µM). We weighed a certain weight of flavonoids, then dissolved the flavonoids with water , or dimethyl sulfoxide  to prepare the stock solutions, and diluted the stock solutions with OptiMEM (Gibco) to the appropriate concentrations.4 cells/well and cultured for 24 h. The medium was replaced with the transfection medium, OptiMEM (Gibco) containing flavonoids and lipoplexes (200 ng/well pcDNA3/GL in Lipofectamine 3000). After 4 h, the cells were washed with phosphate-buffered saline  and incubated with 10% FBS/medium for another 4 h at 37 °C.For 2D cultures, the cells were seeded into a 96-well plate at a density of 1 × 104 cells/well and cultured for 4 h, 3 d, and 7 d. Transfection was performed according to the same protocol used for the 2D cultures.For 3D cultures, the cells were seeded onto a 3D cell culture scaffold Cellbed  in a 96-well plate format at a density of 5 × 10Renilla Luciferase Assay System containing Gaussia luciferase substrate coelenterazine; Promega, Madison, WI, USA), and bioluminescence levels were measured using a luminometer . Luciferase activity is indicated as relative light units (RLU)/mL of medium. After collecting the medium, each well was washed with PBS. Next, 100 µL of medium containing 10 µL of Cell Counting kit-8  was added. The cells were then incubated for 1 h. Absorbance was measured at 450 nm using a microplate photometer . A medium without cells served as a negative control. For 3D cultures, the reactants of CCK-8 were transferred to another 96-well plate to avoid intrinsic absorbance of the scaffold.Transfection efficiency in cells was assessed by mixing 2 µL medium with 20 µL substrate  and incubating for 5 min. The cell suspension was centrifuged at 800× g for 5 min at 4 °C. The cells were washed twice with PBS containing 5% FBS, resuspended in the same solution, and analyzed by flow cytometry .Cells were seeded into a 24-well plate at a density of 8 × 104 cells/cm2 and pre-incubated for 24 h. Cells were washed thrice with PBS and pre-incubated with DALGreen for 30 min, according to the manufacturer’s protocol. After that, the cells were washed thrice with PBS and incubated with OptiMEM containing lipoplexes of pTagBFP-N/Cy5-labeled pTagBFP-N (0.5 µg each), with or without 25 µM quercetin, for 4 h. After washing the cells thrice with PBS, CellROX Orange was added to each dish. After 30 min of incubation, cells were washed thrice again and incubated with a fresh medium containing 10% FBS for a further 12 h. The samples were observed under a confocal laser scanning microscope .HepG2 cells were seeded in 35 mm glass-based dishes at a cell density of 6 × 10p < 0.05.Statistical comparisons were performed using a one-way analysis of variance, followed by Dunnett’s post-hoc test for multiple comparisons with a control group. Statistical significance was set at We tested the effect of co-incubation of flavonoids on the lipofection efficiency in 2D Colon26 cells . All tesNext, we tested the effect of co-incubation with flavonoids on lipofection efficiency in 2D HepG2 cells . A similWe summarized the gene expression promotion effect of flavonoids in descending order . Log P vAlthough 2D cell cultures are still used in most studies, 3D cell culture has a broader range of applications as an analysis technology due to features that are closer to the complex in vivo conditions , proper Three different time points were investigated after cell seeding. The results revealed different gene expression levels. Four hours after seeding, the cells showed the highest gene expression levels compared to the other two timings A. This mIn 3D cultures of HepG2 cells, a trend similar to that in Colon26 cells was observed . Again, Using a flow cytometer, the relationship between ROS levels and gene expression was analyzed in 2D Colon26 and HepG2 cells . There wHere, we selected HepG2 cells for observation by confocal microscopy in the next step.In this study, the correlation between ROS levels and gene expression was consistent with a previous study using edaravone . There mBacterial plasmid DNA can be recognized as foreign material by mammalian cells . ActivatWe only selected a single time point for confocal observation and hypothesized that autophagy during transfection also involves the cell cycle because information on the whole transfection period was missing. As previously mentioned, the nuclear import of plasmid DNA can occur during mitosis. Autophagic flux is highly active during early mitosis ,62. MeanThis study demonstrated the potential of eight flavonoids to increase the transfection efficiency of both Colon26 and HepG2 cells. Quercetin showed the highest promotion effect among tested flavonoids. Optimal concentrations of quercetin were different in 2D (25 µM) and 3D (12.5 µM) cultures. The promotion effect was achieved through scavenging excessive ROS and modulating autophagy to moderate levels, which was induced by cationic lipoplexes. Moderate regulation of ROS and autophagy levels is important for gene delivery. Further studies are needed to obtain more comprehensive information on the entire transfection process.


However, NMR CSP experiments indicate the compounds do not bind specifically to apo- or Mg2+-bound APE1, pointing to a non-specific mode of inhibition, possibly DNA binding. Our results highlight methods for rigorous interrogation of putative APE1 inhibitors and should facilitate future efforts to discover compounds that specifically inhibit this important repair enzyme.AP endonuclease 1 (APE1) processes DNA lesions including apurinic/apyrimidinic sites and 3´-blocking groups, mediating base excision repair and single strand break repair. Much effort has focused on developing specific inhibitors of APE1, which could have important applications in basic research and potentially lead to clinical anticancer agents. We used structural, biophysical, and biochemical methods to characterize several reported inhibitors, including 7-nitroindole-2-carboxylic acid (CRT0044876), given its small size, reported potency, and widespread use for studying APE1. Intriguingly, NMR chemical shift perturbation (CSP) experiments show that CRT0044876 and three similar indole-2-carboxylic acids bind a pocket distal from the APE1 active site. A crystal structure confirms these findings and defines the pose for 5-nitroindole-2-carboxylic acid. However, dynamic light scattering experiments show the indole compounds form colloidal aggregates that could bind (sequester) APE1, causing nonspecific inhibition. Endonuclease assays show the compounds lack significant APE1 inhibition under conditions (detergent) that disrupt aggregation. Thus, binding of the indole-2-carboxylic acids at the remote pocket does not inhibit APE1 repair activity. Myricetin also forms aggregates and lacks APE1 inhibition under aggregate-disrupting conditions. Two other reported compounds  inhibit APE1  N-glycosyl bond (depurination) or by the activity of DNA glycosylases, which initiate BER ) was fitted to 50, the range of FA, and the slope factor (s).Reactions in the presence of a given compound were performed using the same conditions with a DNA substrate concentration of 80 nM and varying concentrations of the compound. In all cases, the compound was introduced from a 100x stock prepared in DMSO, giving a final DMSO concentration of 1%. Samples containing all components (except MgClDynamic light scattering (DLS) experiments were performed to monitor compound aggregation, using a SpectroLight 610 instrument . The instrument features a laser diode (λ 658 nm) which was set to a power of 100 mW and a scattering angle of 150°. For each sample, five replicates of 60 sec duration were collected at 20°C. Instrument settings, including integration time, were held constant for all experiments. Samples (1 ul) were prepared under mineral oil in 96 well SBS plates. Samples included the same buffer used for enzyme activity assays, with or without Brij 35 . The compounds were introduced from 100x stocks prepared in DMSO, giving a final DMSO concentration of 1%; samples without compound contained 1% DMSO. The DLS studies were in some cases performed using compound stocks that had been stored at –20°C in DMSO. Control experiments performed with fresh 100x stocks in DMSO showed that, upon dilution, the compounds formed aggregates at the same concentrations as samples which had been stored at –20°C, indicating that the freeze-thaw process did not cause the observed aggregation.15N-labeled APE1ΔN38 (0.05 mM to 0.15 mM) in 0.02 M sodium phosphate pH 6.5, 0.1 M NaCl, 0.5 mM DTT, 0.2 mM EDTA, 10% D2O. The compounds were dissolved at 100x concentration in deuterated DMSO (DMSO-D6) and were added to the NMR samples at 1% (v/v) to give the desired compound concentration (and 1% DMSO-D6). Control samples contained APE1 with 1% DMSO-D6. For NMR samples that contained MgCl2, concentrated APE1 stocks were dialyzed three times against 1 L of NMR buffer that contained 1 ml of a 50% (w/v) slurry of chelex-100 (Bio-Rad), and the buffer used to prepare NMR samples was similarly treated with chelex-100. 15N-TROSY experiments were collected at 25°C on a Bruker 800 MHz spectrometer equipped with a room temperature probe, as previously reported  to obtain the steady-state (Michaelis-Menten) parameters kcat = 2.2 ± 0.1 s−1 and Km = 76 ± 9 nM  for which the parameter was carefully determined under similar experimental conditions  >0.25 mM, some peaks in the TROSY spectra become weak  or disappear   . Moreove030 ppm) . The resund APE1 , 73. To 030 ppm) . A superexpected . The Mg231, 309)  Figs. As15N-TROSY experiments to investigate whether compounds 6 or 7 bind to the Mg2+-bound form of APE1. We find that compound 6 (0.30 mM) does not induce CSPs (Δδ >0.015 ppm) for any residue of APE1 (0.10 mM) in the presence of MgCl2 at a concentration of 0.25 mM or 1.0 mM , though this seems unlikely.We performed r 1.0 mM . Given og to DNA , which c7 (0.030 mM) also fails to induce CSPs (Δδ >0.015 ppm) for any backbone residue of APE1 (0.05 mM) in the presence of MgCl2 at a concentration of 0.25 mM or 1.0 mM  or to AP-DNA. As noted above, such a mechanism could potentially account for findings (by EMSA) that compound 7, at a concentration of 0.03 mM (as used in our NMR studies), nearly completely disrupts a tight ES complex involving APE1 (28 nM) and AP-DNA (10 nM)  at 0.063 and 0.125 mM. Two residues near the Mg2+-binding site  exhibit peaks for apo APE1 but not APE1 with MgCl2 (≥0.063 mM). (b) CSPs (Δδ) induced by MgCl2 (0.25 mM) as a function of amino acid residue. Labels with one or more stars denote residues for which a peak is not seen in spectra collected for APE1 with 0.25 mM MgCl2; for these residues, Δδ values were calculated using spectra for APE1 with the highest [MgCl2] for which the peak is observed . Residues exhibiting Δδ ≥ 0.015 ppm are labeled in both figures.(a) (TIF)Click here for additional data file.S11 Fig15N-TROSY spectra for APE1 (0.10 mM) in the absence (black) or presence (red) of MgCl2 (1.0 mM). Spectra were also collected for APE1 with lower MgCl2 concentrations . Two residues near the Mg2+-binding site  exhibit peaks for apo APE1 but not APE1 with MgCl2 (≥0.063 mM). (b) CSPs (Δδ) induced by MgCl2 (1.0 mM) versus amino acid residue. Labels with stars mark residues for which a peak is not seen in spectra of APE1 with 1.0 mM MgCl2; for these residues, Δδ values were calculated using spectra for APE1 with the highest [MgCl2] for which that peak is observed . Residues exhibiting Δδ ≥ 0.015 ppm are labeled.(a) (TIF)Click here for additional data file.S12 Fig6 and 0.10 mM APE1 in the presence of MgCl2 at a concentration of (a) 1.0 mM or (b) 0.25 mM.NMR experiments were performed using 0.30 mM compound (TIF)Click here for additional data file.S13 Fig7 and 0.05 mM APE1 in the presence of MgCl2 at a concentration of (a) 1.0 mM or (b) 0.25 mM. The NMR samples also contained detergent (0.05% Brij 35) which helps to preclude aggregation of 7 but does alter the spectra of APE1 in the absence of ligand.NMR experiments were performed using 0.030 mM compound (TIF)Click here for additional data file.S1 TableValues shown in parenthesis are for highest resolution shell. The Ramachandran analysis was performed using Procheck .(DOCX)Click here for additional data file.S1 File(MTZ)Click here for additional data file.S2 File(PDB)Click here for additional data file.S3 File(MTZ)Click here for additional data file.S4 File(PDB)Click here for additional data file.


While EPO induces Ca2+ influx through TRPC2, TRPC6 inhibits the function of TRPC2. Thus, interactions between TRPC2 and TRPC6 regulate the rate of Ca2+ influx in EPO-induced erythropoiesis. In this study, we observed that the expression of TRPC6 in KIT-positive erythroid progenitor cells was regulated by DOT1L. DOT1L is a methyltransferase that plays an important role in many biological processes during embryonic development including early erythropoiesis. We previously reported that Dot1l knockout (KODot1l) HPCs in the yolk sac failed to develop properly, which resulted in lethal anemia. In this study, we detected a marked downregulation of Trpc6 gene expression in Dot1lKO progenitor cells in the yolk sac compared to the wild type (WT). The promoter and the proximal regions of the Trpc6 gene locus exhibited an enrichment of H3K79 methylation, which is mediated solely by DOT1L. However, the expression of Trpc2, the positive regulator of Ca2+ influx, remained unchanged, resulting in an increased TRPC2/TRPC6 ratio. As the loss of DOT1L decreased TRPC6, which inhibited Ca2+ influx by TRPC2, KODot1l HPCs in the yolk sac exhibited accelerated and sustained elevated levels of Ca2+ influx. Such heightened Ca2+ levels might have detrimental effects on the growth and proliferation of HPCs in response to EPO.Erythropoietin (EPO) signaling plays a vital role in erythropoiesis by regulating proliferation and lineage-specific differentiation of murine hematopoietic progenitor cells (HPCs). An important downstream response of EPO signaling is calcium (Ca Amongstchannels ,5,6,7. Tgenitors . It has genitors ,10. Amongenitors  as well genitors ,11. In agenitors ,14,15,16genitors . In erytKODot1l) mice results in an embryonic erythropoietic defect and embryonic lethality during mid-gestation in these mice [KODot1l model showed that DOT1L is also required for murine postnatal hematopoiesis [In eukaryotic cells, DNA is packaged within the nucleus along with histones and other nuclear proteins to form the nucleosome, the fundamental repeating unit of chromatin. The histones can be post-translationally modified in a variety of ways including acetylation, phosphorylation, ubiquitination, and methylation. These modifications influence chromatin structure, facilitate interactions between nucleosomes, and can potentially regulate transcription . Methylaese mice . Consistopoiesis ,28.KODot1l erythroid progenitors failed to develop normally, showing cell cycle arrest and increased apoptosis [Trpc6 is a direct target of DOT1L in these cells. We detected an enrichment of H3K79 methylation within the Trpc6 gene locus. Moreover, the loss of Trpc6 expression in KODot1l HPCs showed accelerated and sustained high levels of Ca2+ influx and correlated with erythroid progenitor cell cycle arrest and death.poptosis . HoweverKODot1l embryos and yolk sacs were markedly smaller than the wild-type embryos and yolk sacs  [KODot1l erythroblasts showed severely blunted proliferation, which resulted in a reduced number of growing cells compared to the wild type (Embryos and yolk sacs were collected on embryonic day 10.5 (E10.5) A. E10.5 olk sacs B,C. Whilere pale B,C. Equa (ESREs) . Dot1lKOild type D,E.KODot1l erythroblasts underwent a G0/G1 cell cycle arrest, which resulted in significantly reduced numbers of cells in the S and G2/M phases  colonies than those of the wild type (KODot1l myeloid (CFU-GM) colonies were similar in size , only Trpc6 levels were significantly reduced in KODot1l progenitor cells. Other TRPC members remained relatively unchanged  [Trpc6 locus    [KODot1l embryos [We observed that the first wave of primitive as well as the second wave of definitive HPCs were present in the olk sacs ,30. Altholk sacs ,30. Despignaling ; howeverignaling ,33. An aes (ROS) ,35 and ces (ROS) ,37, whic embryos ,30.KODot1l yolk sacs underwent cell cycle arrest and apoptosis. The cultured HPCs failed to expand  compared to those that expressed lower amounts   . We also2+ influx [KODot1l erythroid progenitor cells, while others, including TRPC2 levels, remained intact, resulting in a four-fold increase in the TRPC2/TRPC6 ratio. Previous studies have shown that interaction between TRPC2 and TRPC6 is essential for modulating EPO-induced Ca2+ signaling [2+ entry in KODot1l erythroid progenitor cells , 2 U/mL human recombinant EPO , 100 ng/mL SCF , 10 µM dexamethasone , 40 ng/mL IGF1  and penicillin–streptomycin  [Single-cell suspensions of E10.5 wild-type and MA, USA)  as descrMA, USA) . After 4MA, USA) ,26. SepaMA, USA) ,26. FlowMA, USA) ,26 at thKODot1l yolk sacs were plated in 35 mm culture dishes in M3434 methylcellulose medium  containing cytokines SCF (100 ng/mL), EPO (2 U/mL), IL-3 (5 ng/mL), and IL-6 (5 ng/mL) , which promoted definitive erythroid and myeloid colony formation [Definitive erythropoiesis, erythroid (BFU-E), and myeloid (CFU-GM) colony formation assays were performed as described previously . Approxiormation . The celSingle-cell suspensions were prepared from E10.5 yolk sacs as described above, and KIT-positive cells were separated as reported previously ,26. Briehttp://pga.mgh.harvard.edu/primerbank/, accessed on 1 May 2011).Total RNA was extracted using TRIzol reagent  following the manufacturer’s instructions. cDNAs were prepared from 1 µg of total RNA collected from each sample using the SuperScript VILO cDNA Synthesis Kit . Quantitative real-time PCR (qPCR) was performed using Power SYBR Green Master Mix  and ran on a 7500 real-time PCR system . qPCR results were normalized to Rn18S expression and calculated by the comparative ΔΔCT method ,41,42. Ag at 4 °C for 20 min. Supernatants were collected and protein concentrations were measured using the Bradford assay . Approximately 30 µg of each protein sample was mixed with NuPAGE SDS 4× sample buffer  and were electrophoresed on a 10% precast gel in SDS running buffer  . Proteins were transferred to PVDF membranes, and the membranes were blocked in 5% bovine serum albumin (BSA) overnight at 4 °C. Blocked membranes were incubated with 1:300 TRPC6 primary antibody  for 4 h at 4 °C with shaking. Membranes were washed 5 times with TBST buffer at 4 °C with shaking and incubated with 1:5000 secondary antibody (Goat anti-Mouse IRDye CW800) for 45 min at room temperature. Then, the membranes were washed another 5 times with TBST, and images were captured with an Odyssey CLx infrared image system . The same membrane was stripped and incubated with an antibody against ACTB  as a loading control of the protein samples.Western blot analyses of TRPC6 were performed following standard protocols. Cells from the whole yolk sac, bone marrow, or liver were lysed in Nonidet-P40 (NP-40) buffer  on ice for 30 min. Cell extracts were centrifuged at 12,000× Trpc6 [Bone marrow cells that expressed high levels of Trpc6  were isoTrpc6 . Briefly7 cells were resuspended in 10 mL IMDM medium containing 5% FBS and were fixed in 1% formaldehyde for 10 min. Glycine was added to a final concentration of 125 mM and incubated at room temperature with rotation for 10 min to neutralize formaldehyde. The cells were washed twice with cold PBS, lysed in 500 µL SDS lysis buffer with protease inhibitor cocktail , and incubated on ice for 10 min. The nuclear pellets were washed with a cold lysis buffer and sonicated using a VWR Branson Sonifier 250 probe sonicator  4–6 times, 15 s each time, to reduce the chromatin length to between 200 and 1000 bp. Sonicated chromatin solutions were centrifuged at 10,000× g at 4 °C for 10 min. Clear supernatants were collected into 1.5 mL tubes, and ~500 µL chromatin solution was mixed with 75 µL Protein A Agarose/Salmon Sperm DNA and incubated at 4 °C with rotation overnight. The solution was centrifuged at 3000× g for 5 min. The supernatant was transferred to a new tube and diluted 10-fold in ChIP Dilution Buffer. Two micrograms of H3K79me2 or H3K79me3 antibody  was added to 1 mL of the diluted chromatin solution each, and another 1 mL was used as “input”. Two micrograms of normal rabbit IgG  was used as negative control.Chromatin immunoprecipitation (ChIP) was carried out following standard protocols ,44. Brieg for 5 min at 4 °C, and the supernatants were discarded. The Protein A Agarose beads were washed for 5 min on a rotating platform sequentially with 1 mL of each of the low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, and 1XTE. Then, the chromatins were eluted with 250 µL elution buffers twice, and the elutes were combined. Twenty microliters of 5 M NaCl and 2 µL RNase were added to each eluted chromatin sample and incubated at 37 °C for 30 min to remove RNAs and then again at 65 °C for 4 h to decrosslink the ChIPed chromatin materials. The chromatin solutions were mixed with 10 µL of 0.5 M EDTA, 20 µL 1 M Tris-HCl, PH 6.5, and 2 µL of 10 mg/mL Proteinase K and incubated at 45 °C for 1 h. Finally, the chromatin DNA fragments were recovered by phenol/chloroform extraction and ethanol precipitation. Real-time PCR was performed to quantify precipitated DNA with the use of the 7500 real-time PCR system. The ChIP-qPCR primers used for analyzing the Trpc6 locus are listed in Chromatin solutions were incubated at 4 °C with rotation for 4 h in the presence of specific antibodies. Then, 65 µL Protein A Agarose/Salmon Sperm DNA was added to each tube and incubated at 4 °C with rotation for another 2 h. The solutions were centrifuged at 3000× 2+ concentration change of HPCs when treated with EPO. Fura2 was used as the detection fluorophore. After the whole yolk sacs were dissociated into single cells, the cells were washed 2–3 times with PBS to completely remove fetal bovine serum (FBS). HPCs were resuspended in 40 µL RPMI 1640  without FBS. Cells were loaded into the center of a culture dish coated with poly-lysine and fixed for 10 min at room temperature. The medium was then removed, and the cells were stained with 1 µM Fura2 and Pluronic F127  (1:1 in volume) at 37 °C for 30 min. The staining solution was removed, and the cells were covered with 150 µL RPMI containing 10% FBS. Mouse EPO  was added to a final concentration of 10 units/mL. Then, the sample was analyzed immediately by confocal fluorescence microscopy on a Nikon TE2000U microscope . Fura2-loaded cells were visualized by digital video imaging, and the fluorescence was quantitated using the intensity ratio of the emission (510 nm), which was measured following excitation at 340 nm divided by the emission following excitation at 380 nm. Each sample was measured over 20 min. After, the data were calculated and analyzed using Metamorph 6.1 .A fluorescence microscopy-coupled digital imaging system was used to measure the Cap < 0.05. All the statistical calculations were performed using SPSS 22 .Each experimental group consisted of a minimum of 6 samples. The experimental results are presented as the mean ± standard error (SE). The results were analyzed for one-way ANOVA, and the significance of the mean differences was determined by Duncan’s post hoc test with


However, published pharmacological and structural data remain controversial. Here, we investigated the molecular pharmacology of apamin by two-electrode voltage-clamp in Xenopus laevis oocytes and patch-clamp in HEK293, COS7, and CHO cells expressing the studied ion channels, as well as in isolated rat brain neurons. The microtitre broth dilution method was used for antimicrobial activity screening. The spatial structure of apamin in aqueous solution was determined by NMR spectroscopy. We tested apamin against 42 ion channels  and confirmed its unique selectivity to KCa2 channels. No antimicrobial activity was detected for apamin against Gram-positive or Gram-negative bacteria. The NMR solution structure of apamin was deposited in the Protein Data Bank. The results presented here demonstrate that apamin is a selective nanomolar or even subnanomolar-affinity KCa2 inhibitor with no significant effects on other molecular targets. The spatial structure as well as ample functional data provided here support the use of apamin as a KCa2-selective pharmacological tool and as a template for drug design.Apamin is often cited as one of the few substances selectively acting on small-conductance Ca Moreoveodotoxin . A plethodotoxin .Apis mellifera and purified a minor peptide component that he named “apamin” . The symptoms are apparently of central origin because the toxicity increases by a factor of 1,000–10,000 and the progression of poisoning is faster in case of i.c.v. injection, but the clinical picture stays the same .Studies of the spatial structure of apamin began as early as the late 1970s, but no atomic coordinates have been deposited in the Protein Data Bank. The first NMR study provided an erroneous assignment of the secondary structure elements , which w2+-activated K+ ion channels (KCa)  and not intermediate-conductance (KCa3.1) or large-conductance (KCa1.1) KCa channels.  and less potent on KCa2.1  (Ca2.3 (SK3 or SKCa3) channels showed an intermediate sensitivity (IC50 = 0.6–4.0 nM)  . Since thannels. . After t targets . A numbe7–12 nM) , whereas–4.0 nM) .2+ and/or Na+ channels in the embryonic chicken heart  and synthetic melittin (M4171) purchased from Sigma-Aldrich. TRAM-34 was synthesized as described  and are permanently archived in the Concise Guide to PHARMACOLOGY 2021/22  (Ion channel targets and their ligands are presented according to IUPHAR/BPS Guide to PHARMACOLOGY ( 2021/22 . For potmdb.org) .18 column  in a linear gradient of acetonitrile concentration (0%–60% in 60 min) in the presence of 0.1% trifluoroacetic acid (TFA). 1525 Binary HPLC pump and 2489 UV/Visible detector under the control of Breeze 2 software  were used for chromatography.Apamin purity was confirmed using reversed-phase (RP) HPLC on a Vydac 218TP54 CMolecular mass measurements were performed using MALDI on an Ultraflex III TOF-TOF instrument (Bruker). 2,5-Dihydroxybenzoic acid (Sigma-Aldrich) was used as a matrix. Measurements were carried out in the reflector mode, which enabled isotopic resolution. Calibration was performed using the ProteoMass Peptide MALDI-MS Calibration Kit (Sigma-Aldrich). Mass spectra were analyzed with the Data Analysis 4.3 and Data Analysis Viewer 4.3 software (Bruker).Apamin and melittin concentrations were determined by UV spectrophotometry. To obtain absorption spectra of those substances in the UV range, lyophilized peptides were dissolved in 0.5 ml of water (Milli-Q produced on a Merck Millipore Water Purification System). We used a UV-1800 spectrophotometer (Shimadzu) and quartz cuvettes with an optical path length of 1.0 cm; water served as a reference solution. Apamin does not contain aromatic amino acid residues; therefore, absorbance at 205 nm was used to measure the concentration . MelittiEnterococcus faecalis ATCC 29212, Staphylococcus aureus subsp. aureus ATCC 29213) and Gram-negative bacteria  following the previously described modification of the microtitre broth dilution method . The microtitre plates were incubated for 24 h at 37°C, and growth inhibition was determined by measuring the absorbance at 620 nm. Minimum inhibitory concentration (MIC) is expressed as the lowest concentration of peptide that caused 100% bacterial growth inhibition.Briefly, bacteria were cultured in a low-salt LB medium. The two-fold microtitre broth dilution assay was carried out in 96-well sterile plates in a final volume of 100 μL. Mid-exponential-phase cultures were diluted to a final concentration of 102O/D2O mixture (95:5) and placed into a 5-mm Shigemi NMR tube. The sample pH was adjusted to 3.2. NMR spectra of apamin were recorded using a 600-MHz Bruker Avance III NMR spectrometer, equipped with a triple-resonance cryogenic probe, at 25°C. We recorded the following spectra: DQF-COSY, NOESY (120 ms), ROESY (200 ms), TOCSY (80 ms), 13C and 15N-HSQC at natural abundance. 3JHNHA were measured by the line shape analysis of cross-peaks in NOESY spectra, whereas 3JHAHB couplings of AMX spin systems were determined by the line shape analysis of cross-peaks in 2D TOCSY spectra.For NMR studies 1 mg of apamin was dissolved in 320 μL of H3D structure calculation was performed using the simulated annealing/molecular dynamics protocol as implemented in the CYANA software package version 3.98 . The disXenopus oocytes: for voltage-gated potassium channels, KV , inward-rectifier potassium channels, Kir [mKir3.1/3.2 or GIRK1/2 (NM_008426/XM_011246104)], voltage-gated sodium channels, NaV , transient receptor potential channels, TRP , nicotinic acetylcholine receptors, nAChR .The following genes encoding ion channel subunits were expressed in X. laevis frogs was described previously (−1 using a microinjector (Drummond Scientific). The oocytes were incubated at 16°C in ND96 solution containing (in mM): NaCl, 96; KCl, 2; CaCl2, 1.8; MgCl2, 2; and HEPES, 5 (pH 7.4), supplemented with 50 mg l−1 gentamicin sulfate.Linearized plasmids bearing the ion channel genes were transcribed using the mMESSAGE mMACHINE SP6 or T7 transcription kits (Ambion) to prepare the respective cRNA. The harvesting of stage V–VI oocytes from anaesthetized female eviously . OocytesCa1.1 , hKCa2.1 , and hKCa3.1  or COS7 cells stably expressing hKCa2.3  were generated and cultured as previously described  were a gift from Dr. Mio Zhang .HEK293 cells stably expressing hKescribed . HEK293 2 incubator (Binder) at 37°C in a humidified atmosphere of 5% CO2. Cells were maintained under standard culture conditions  in 35 mm2 Petri dishes. Transfection was performed using 0.5 μg of plasmids encoding rASIC1a (NM_024154), rASIC2a (NM_001034014), or rASIC3 (NM_173135) gifted by Dr. Alexander Staruschenko  with 0.5 μg of a GFP-encoding plasmid and Lipofectamine 2000 (Invitrogen) according the manufacturer’s protocol. Patch-clamp experiments were performed 48 h after transfection.CHO cells were used for transient expression of acid-sensing ion channels (ASIC). These cells were cultured in a CB-150 CO2, 1.3; MgCl2, 2; NaHCO3, 26; NaH2PO4, 1.24; and D-glucose, 10; aerated with carbogen . Transverse slices, comprising hippocampus and striatum, were cut with a 7000 SMZ-2 vibratome (Campden Instruments) and stored at room temperature (22°C–24°C) in ACSF aerated with carbogen. Neurons were isolated from the slices by vibrodissociation  were deeply anaesthetized with isoflurane and sacrificed by cervical dislocation followed by decapitation. The brains were quickly removed and immersed in ice-cold (2°C–4°C) artificial cerebrospinal fluid (ACSF) of the following composition (in mM): NaCl, 124; KCl, 5; CaClociation . The effociation  and Ca2+eceptors  and on geceptors . The expteromers .2, 1.8; MgCl2, 2; and HEPES, 5 (pH 7.4). Voltage and current electrodes were filled with 3 M KCl. Resistances of both electrodes were kept at 0.7–1.5 MΩ. Elicited currents were sampled at 1 kHz and filtered at 0.5 kHz (for potassium currents) or sampled at 20 kHz and filtered at 2 kHz (for sodium currents) using a four-pole low-pass Bessel filter. Leak subtraction was performed using a −P/4 protocol.Recordings were performed at room temperature (18°C–22°C) using a Geneclamp 500 amplifier (Molecular Devices) controlled by a pClamp data acquisition system (Axon Instruments). Whole-cell currents from oocytes were recorded 1–4 days after cRNA injection. Bath solution composition was ND96, or HK containing (in mM): NaCl, 2; KCl, 96; CaClV) or 500 ms (KV) depolarization to the voltage corresponding to the maximal activation of the channels in control conditions from a holding potential of −90 mV. For nAChR experiments, the oocytes were voltage-clamped at a holding potential of −70 mV and continuously superfused with ND96 via gravity-fed tubes at 0.1–0.2 ml min−1, with 5 min incubation times for the bath-applied peptides. ACh was applied via gravity-fed tubes until peak current amplitude was obtained (1–3 s), with 1–2 min washout periods between applications. The nAChR were gated by a particular time duration pulse of ACh for the respective nAChR subtype  at 2 ml min−1. Data were sampled at 500 Hz and filtered at 200 Hz. TRP currents were measured in ND96 at −90 mV during 400 s. Capsaicin (2 μM) was used as an agonist and capsazepine (10 μM) as an antagonist of TRPV1. Peak current amplitude was measured prior to and following the application of the peptide. All data were obtained in at least five independent experiments (n ≥ 5).Currents were evoked by a 100 ms . For current measurements, we used an internal pipette solution containing (in mM): K+ aspartate, 160; MgCl2, 2.08; HEPES, 10; EGTA, 10; and CaCl2, 8.55 (1 μM free Ca2+); pH 7.2; osmolarity 310 mOsm. Free Ca2+ concentrations were calculated with MaxChelator  assuming a temperature of 25°C, pH 7.2, and ionic strength of 160 mM. Na+ Ringer was used as an external solution containing (in mM): NaCl, 160; KCl, 4.5; CaCl2, 2; MgCl2, 1; and HEPES, 10; pH 7.4; osmolarity, 315 mOsm. Please note that the provided concentrations are what was weighed in to achieve an initially hyperosmolar solution that was diluted with water to 310 mOsm for the internal and 315 mOsm for the external solution as measured with a VAPRO Vapor pressure osmometer (Wescor). A slight 5 mOsm difference in osmolarity between internal and external solution improves the sealing rate.All experiments were conducted with an EPC-10 amplifier (HEKA) in the whole-cell configuration with a holding potential of –80 mV. Pipette resistances averaged around 2.5 MΩ. Solutions of apamin in NaCa2 and KCa3.1 currents were elicited by 200-ms voltage ramps from –120 to 40 mV applied every 10 s, and the fold decrease of slope conductance at –80 mV was taken as a measure of apamin-induced channel inhibition. TRAM-34 was used to block KCa3.1. KCa1.1 channel activity was recorded with a step protocol, in which cells were stepped to +40 mV for 100 ms at 30-s intervals. Concentration-dependent current inhibition was fitted with the Hill equation using Prism 8 (GraphPad Software). All data were collected in at least five independent experiments (n ≥ 5).K2, 2.5; D-glucose, 10; and HEPES, 10; and the pH was adjusted to 7.4 with HCl. 10 mM MES was used in the extracellular solution in experiments on ASIC. The patch pipettes (2.5–3.5 MΩ) were made from borosilicate glass (WPI) using a P-97 puller (Sutter Instruments). The pipette solution contained (in mM): CsF, 100; CsCl, 40; NaCl, 5; CaCl2, 0.5; EGTA, 5; and HEPES, 10; and the pH was adjusted to 7.2 with CsOH. All experiments were performed at room temperature (22°C–24°C). NMDA receptors were activated by 100 μM NMDA and 10 μM glycine. AMPA receptors were activated by 100 μM kainate. ASIC of rat neurons were activated by pH drops from pH 7.4 to 6.5. ASIC1a, ASIC2a, and ASIC3 homomers were expressed in CHO cells and activated by pH 6.5, 5.0, and 6.8, respectively. Lyophilized apamin was freshly dissolved in extracellular solution prior to the experiment. After recording the control response, the patched cell was superfused for 40 s with extracellular solution containing apamin, and then the response to co-application of the agonist and apamin was recorded. Offline data analyses were performed using Origin 9.1 (OriginLab) software. All data were collected from at least five experiments (n ≥ 5).In this case, the holding potential was set at –70 mV. Signals were filtered at 10 kHz and sampled at 20 kHz. Drugs were applied using an RSC-200 (BioLogic) perfusion system. The extracellular solution contained (in mM): NaCl, 143; KCl, 5; CaClCa channels are means ± standard deviation (SD) from at least five independent experiments. IC50s are reported with 95% confidence intervals (CI). The CI is based on the fit for the averaged currents. Student’s two-tailed unpaired t-test was used to assess the significance of the effect of apamin in rat neurons (current in the presence of apamin vs current in control). A value of p < 0.05 was considered statistically significant.Data and statistical analysis comply with recommendations on experimental design and analysis in pharmacology . All dat+ = 2026.7 Da; Δ = 0.2 Da).Natural apamin isolated from bee venom was purchased from Sigma and subjected to analytical RP-HPLC. It presented a single symmetrical Gaussian peak  correspoAs there is no apamin structure in the PDB, we solved it with NMR spectroscopy in solution based on the recorded J-couplings and NOE distances . NMR cheApamin forms a short ɑ-helix (Ala9–Gln16) and a β-turn  channels  or large-conductance KCa1.1 (BK) currents at 5 μM  and ASIC were studied in isolated neurons of rat brain. Extracellular application of apamin alone did not produce noticeable effects on the holding currents. We compared the whole-cell currents through the receptors in control and in the presence of apamin. At a concentration of 5 µM, it produced no effects on the three types of iGluR and ASIC. In the case of NMDA receptors we noticed that the current in the presence of apamin tended to be slightly lower than in control (in some cells up to ≈15%), although this difference was not statistically significant  are a group of three α-subunit isoforms (KCa2.1–2.3) that can form mature channels of either homo- or heterotetrameric structure . Unlike KV channels or large-conductance Ca2+-activated K+ channels (BK or KCa1.1), the gating of SK channels is insensitive to transmembrane voltage. KCa2 channels have no Ca2+ binding sites in their α-subunit, but they form a stable complex with calmodulin that acts as their Ca2+ sensor   , KCa2.1 Ca2 with high (nanomolar) affinity is limited. According to the Kalium database   resulted in derivatives with enhanced selectivity for KCa2.2 over KCa2.3 (The number of known polypeptide or peptide toxins acting on Kmdb.org) , six of hebraeus  and tama tamulus , in partr KCa2.3 . The impCa2.2 channel pore region, whereas Gln17 interacts with Asn368  (Ca2.2 and KCa2.3 (Q9UGI6), respectively, and Asn345 of KCa2.2; all in S5–P] (Ca2.1 (Q92952) sensitivity towards apamin  was implicated in forming the binding interface for apamin  and Gln17 of apamin are key determinants of its activity . Mutagen P70604) . Two addin S5–P] . Those ss apamin . In addir apamin . In the Ca2. Indeed, since the early 1980s apamin has been used as the main pharmacological agent to distinguish KCa2 channels from other K+ channels (Ca2 confirmed that these three channel isoforms are the molecular targets of apamin (2+ and Na+ channels in embryonic heart tissues (V1.3 as a target (+ channels nor KV1.3.It is apamin from the honeybee venom  that is channels . Subsequf apamin . However tissues , 1992. Aa target . Our ext


Solute carrier family 7 member 2 (SLC7A2), a cationic amino acid transporter, is lowly expressed in ovarian and hepatocellular cancers, which is associated with their worse prognosis. However, its roles in the prognosis, drug resistance and immune infiltration in non-small-cell lung cancer (NSCLC) are unclear.We chose SLC7A2 from RNA-Seq of paclitaxel/cisplatin-resistant A549 cells, then bioinformatics, cell lines construction, RT-qPCR, and CCK8 were performed to investigate SLC7A2 role.We analyzed the 223 differentially expressed genes (DEGs) from RNA-Seq of paclitaxel/cisplatin-resistant A549 cells and found that SLC7A2 expression was down-regulated in NSCLC. Lower SLC7A2 expression was associated with worse recurrence-free survival (RFS) in NSCLC. SLC7A2 silencing enhanced the proliferation of NSCLC cells and their insensitivity to paclitaxel, cisplatin, and gemcitabine in vitro. Activation of AMPK has up-regulated SLC7A2 expression and enhanced the sensitivity of NSCLC cells to anti-tumor drugs, which could be attributed to E2F1’s regulation. In addition, the levels of SLC7A2 expression were correlated to the numbers of infiltrated neutrophils, macrophages, dendritic cells and their marker genes, like CD86, HLA-DPA1 and ITGAM.SLC7A2 may act as a tumor suppressor to modulate drug sensitivity, immune infiltration and survival in NSCLC.Video abstractThe online version contains supplementary material available at 10.1186/s12964-022-01023-x. Lung cancer still has the highest mortality rate worldwide . Non-smaThe solute carrier family 7 member 2  is a member of the solute carrier superfamily. Currently, SLC7A2 can functionally transport cationic amino acids, like L-arginine, into the cytosol and regulate inflammation . Recent In this study, we analyzed transcriptomes of wild-type and drug-resistant A549 cells by RNA-seq and explored SLC7A2 expression profiles and associated recurrence-free survival (RFS) of NSCLC patients in available databases and patient’s tissues. Subsequently, we tested the potential functions and mechanism of SLC7A2 in the proliferation and drug sensitivity of NSCLC cells and how activation of AMPK modulated SLC7A2 expression. Finally, we explored the potential association between the levels of SLC7A2 gene transcripts and immune infiltration in NSCLC.Special reagents used in this study included paclitaxel, cisplatin, fluorouracil, gemcitabine, methotrexate vinblastine and Cell Counting Kit-8 (CCK-8) , oxaliplatin and saracatinib ; anti-SLC7A2, anti-β-actin , anti-phosphorylated AMPK, anti-AMPK ; the shRNA-SLC7A2 and control plasmids ; the RNeasy Mini Kit and QIAGEN Nova SYBR Green PCR Kit , and FastKing RT Kit (with gDNase) .The RNA sequencing assay was performed by Gene Denovo Biotechnology . Total mRNAs were extracted from each cell line using Trizol reagent kit , according to the manufacturer’s protocol . The RNA quality was evaluated by Agilent 2100 Bioanalyzer  and RNase free agarose gel electrophoresis. The total mRNAs were further enriched by Oligo(dT) beads  and fragmented into short fragments (about 200 nucleotides) using fragmentation buffer. Subsequently, the fragmented mRNAs were reverse-transcribed into cDNA using random primers, DNA polymerase I, RNase H, dNTP and buffer. After purification, repairing, ligating the cDNA with the Illumina sequencing adapters, agarose gel electrophoresis and PCR assay, the products were sequenced using Illumina Novaseq 6000 .SLC7A2 mRNA transcripts in various types of cancer and normal tissues were analyzed by Oncomine database (https://www.oncomine.org) [p ≤ 0.001 and fold change = 1.5.The relative levels of ine.org)  and the https://www.omicsmart.com/) and three ontologies of molecular function (MF), cellular component (CC) and biological process (BP) for GO analyses. The potential enriched pathways of DEGs were analyzed by KEGG.The potential functions of DEGs were analyzed by GO functional enrichment analyses using Omicshare Online tools (http://dna00.bio.kyutech.ac.jp/PrognoScan/).The correlation between SLC7A2 expression and the RFS of patients with different types of cancers was analyzed using PrognoScan database (https://cistrome.shinyapps.io/timer/) [p ≤ 0.05.The immune infiltrates in various types of cancers were analyzed using TIMER Database (/timer/) . The cor/timer/) , 14. TheTo generate lentivirus, 293 T cells were transfected with pGV654-shRNA-SLC7A2 or Control plasmid, together with lentivirus packaging plasmids using lipofectamine 2000 for 48 h. Their supernatants were collected and filtered through 0.22 mm Millipore filters, followed by ultracentrifuge. NSCLC cells were transduced with each type of lentivirus at MOI of 5 in the presence of polybrene and treated with 500 µg/ml G418 to establish stable SLC7A2-silenced cell lines.6 A549 cells were seeded into one 10 cm dish. Next day, cells were then treated with drug of specified concentration for 15 days. If the cells grew well, we then added the drug with further elevated concentration; if over half of the cells were killed, we should stop drug usage until the number of cells regain 3 × 106. Following this order, we added cisplatin with concentrations of 10, 20, 40, 50, 60, 70, 80, 90 µM. Cisplatin powder was dissolved with dimethylformamide, resulting in 10 mM cisplatin stock solution. And the concentration of paclitaxel is 10, 20, 30, 40, 60, 70, 90, 100 nM. The paclitaxel powder was dissolved with dimethylsulfoxide and the stock solution concentration is 10 µM.3 × 10Drug-sensitive and resistant A549 cells (1500 cells/well) were cultured in triplicate in 96-well plates overnight and treated with different concentrations of individual drugs at a specific concentration for varying time periods. During the last one-hour culture, each well was added with 10 µl of CCK-8. The absorbance at 450 nm in individual wells was measured using a microplate reader.Drug-sensitive and resistant A549 cells (500 cells/well) were cultured in 6-well plates, and treated with, or without, metformin (10 mM) for 15 days. The formed clones were stained with Wright and Giemsa solution and photoimaged.The cells were harvested and lysed in RIPA lysis buffer. After determination of protein concentrations, the cell lysate samples (20 µg/lane) were separated by 10% SDS-gel electrophoresis, and transferred onto PVDF membranes. The membranes were blocked with 5% no-fat dry milk for 1 h and incubated with primary antibodies overnight at 4 °C. Subsequently, the bound antibodies were detected with second antibodies for 1 h at room temperature and visualized using FluorChem HD2 .−ΔΔCT.Total mRNA was extracted from each type of cells and reverse-transcribed into cDNA using RNeasy Mini Kit, according to the manufacturer’s instructions. The relative levels of targeted gene mRNA transcripts to the control GAPDH were quantified by RT-qPCR using SYBR Green Master, specific primers, and cDNA template. The specific primers were forward 5′–CCCGGGATGGCTTACTGTTT–3′ and reverse 5′–CAAAGCAGAAATGACCCCTGC–3′ for SLC7A2 (a product of 95 bp); forward 5′–CAGGGCTGCTTTTAACTCTGGT–3′ and reverse 5′–GATTTTGGAGGGATCTCGCT–3′ for GAPDH (a product of 199 bp), forward 5′–AGTACCTGAGCTCGCCAGT–3′ and reverse 5′–AAGCCCTTGCTGTAGTGGTG–3′ for interleukin 1 beta , forward 5′–TCCCCTGAGGCATTTAGGCA–3′ and reverse 5′–GAAAAGGCTCCCAGGGCTAA–3′ for toll like receptor 4 , forward 5′–ACCTGGGAAATTCAAGGCGT–3′ and reverse 5′–CCGAAGTGCAGATTCCCTCC–3′ for complement C5 , forward 5′–CAGACCACGCAAGGAGTTCA–3′ and reverse 5′–CTTCCACCTTGGAGCACTGT–3′ for C-X-C motif chemokine ligand 5 , forward 5′–GAGGCTCTGAAGGTCCCCA–3′ and reverse 5′–GGGCCAGCAGCACTAGC–3′ for collagen type I alpha 1 chain , The amplification reactions were performed in duplicate and subjected to 40 cycles of 95 °C for 2 min, 95 °C for 5 s, 60 °C for 10 s,. The results were calculated by 27 H460 cells were extracted by 2 ml cell lysis buffer for Western and IP , and then kept 100 µl as pre-IP, 20 µl primary SLC7A2 antibody was incubated with 10 µl protein A/G beads at 4 °C for 4 h. Next, we added the rest protein into antibody- A/G beads overnight at 4 °C. The next day, the protein- antibody-A/G beads were centrifuged at 12,000 g for 5 min. The supernatant was post-IP and the deposit was IP. At last, the E2F1 and SLC7A2 protein were detected by Western blot.The protein of 2 × 10The IHC assay was performed as described before : brieflyt-test, one-way ANOVA using the GraphPad Prism software, Chi-square using the SPSS statistics. A p-value of < 0.05 was considered significant difference.All the data are expressed as mean ± SEM. The difference between groups was analyzed by Student’s Drug resistance is a common reason for chemotherapy failure. To explore the potential mechanisms underlying drug resistance, we established paclitaxel-resistant A549-PTX-R as a previous report  and cispTo understand the potential mechanisms underlying the development of drug resistance, we analyzed the difference in transcriptomes between wild-type and drug-resistant A549 cells by RNA-seq. There were 43,423,127 ± 4,756,524 clean raw reads from cisplatin-resistant cells, 66,035,803 ± 2,190,052 raw reads from paclitaxel-resistant cells and 51,206,167 ± 4,826,313 from A549 wildtype cells. There were 1107 DEGs between paclitaxel-resistant and WT cells. Among them, 447 genes were upregulated and 660 downregulated  members are crucial for transporting anti-tumor drugs into cells and lower SLC expression is associated with drug resistance. Accordingly, we analyzed the expression levels of SLC members and found that SLC7A7, SLC7A2, SLCO4A1, SLC1A3 and SLC44A5 were expressed differentially in both resistant cells. RT-qPCR displayed that compared with that in the WT cells, significantly higher levels of SLC44A5, but lower SLC7A7, SLC7A2, SLCO4A1, SLC1A3 mRNA transcripts were detected in both drug-resistant cells . There were 493 patients with survival information among 501 NSCLC patients (427 were higher SLC7A2 expression and 66 were lower expression). By analyzing with K-M method of ‘survminer’ in the R package to generate the survival curves, we found that patients with higher SLC7A2 expression showed better prognosis   are the most common factors for chemoresistance in tumor cells. Indeed, targeting P-gp by a tyrosine kinase inhibitor pyrazolo [3,4-sistance . Similarsistance . Moreovesistance . In this−/− mice display higher levels of proinflammatory chemokines, like CXCL1 and CXCL5, but down-regulated CXCL9 and IL-4 in the tumors, accompanied by increased pro-tumorigenic M2 macrophage activation [SLC7A2 is a membrane protein and can transport cationic amino acids, like lysine arginine, into cells. Previous studies have demonstrated that lower SLC7A2 expression is associated with worse prognosis of ovarian cancer and hepatocellular carcinoma. In addition, SLC7A2 can down-regulate the expression of CXCL1 to inhibit the infiltration of myeloid-derived suppressor cells and the immune escape of hepatocellular cancer . In conttivation . In addiTo understand the biological roles of SLC7A2, we generated SLC7A2-silenced A549-shRNA-SLC7A2 and H460-shRNA-SLC7A2 cells and found that SLC7A2 silencing promoted the proliferation of NSCLC cells, suggesting SLC7A2 may be a tumor suppressor in NSCLC. On the other hand, we found that SLC7A2 silencing decreased the sensitivity to 5-FU, cisplatin, paclitaxel, methotrexate, vianblastine and gemcitabine in A549-shRNA-SLC7A2, and to cisplatin, paclitaxel, methotrexate, and gemcitabine in H460-shRNA-SLC7A2 cells, respectively. Interestingly, treatment with each drug for two weeks reduced the expression of SLC7A2 in H460 and A549-WT cells, which may stem from drug-resistance of these cells following long-term exposure. Therefore, SLC7A2 may be crucial for prevention and inhibition of chemoresistance in NSCLC. And the E2F1 could be the important upstream transcription factor in regulating the function of SLC7A2 in drug resistance.Our previous gene array results indicated that metformin treatment up-regulated SLC7A2 expression. Indeed, we found that treatment with metformin inhibited the clone formation of both wild-type and drug-resistant NSCLC cells and additionally enhanced the cytotoxicity of cisplatin against NSCLC cells. Such data indicated that activation of AMPK by metformin re-sensitized NSCLC cells to inhibition of cisplatin. Consistently, we found that treatment with metformin or another AMPK activator AICAR up-regulated SLC7A2 expression in NSCLC cells. These results indicated that activation of AMPK attenuated chemoresistance in NSCLC partially by up-regulating SLC7A2. AMPK is an energy sensor and acts as a tumor suppressor . It can Given that immune infiltrates and their specific types and functions are critical for the prognosis of NSCLC, we analyzed the association of SLC7A2 expression with immune infiltrates in NSCLC. We found that the levels of SLC7A2 mRNA transcripts were correlated with the degrees of macrophage, neutrophil and dendritic cell infiltrates in both LUAD and LUSC. Furthermore, the levels of SLC7A2 mRNA transcripts were positively correlated with the levels of genes for macrophage and dendritic cells in LUAD and LUSC. It is notable that SCL7A2 can shift macrophage activation to pro-tumorigenic M2 during the process of colon tumorigenesis . ConsistWe recognized that our study had limitations. First, although we found that lower SLC7A2 expression was associated with worse prognosis of NSCLC, and its deficiency aggravated chemoresistance, we did not determine whether and how SLC7A2 could modulate the influx transporter of multiple anti-tumor drugs. Second, we only tested the potential function of SLC7A2 in regulating the proliferation, chemosensitivity and clone formation of NSCLC cells. However, we did not analyze the functions of other four SLC members that were differentially expressed in drug-resistant A549 cells. In addition, we found that the levels of SLC7A2 gene transcripts were correlated to the degrees of macrophage, neutrophil and dendritic cell infiltrates and their markers, but on one hand, we only concluded it by bioinformatics and predicted CXCL5 and IL-1β genes were related to immune infiltrate, without animal experiments, which is much more straightforward. Therefore, further investigations on the molecular mechanism underlying the roles of SLC7A2 and other SLC members in regulating the growth, chemoresistance and immune infiltration are warranted in future studies.SLC7A2 may act as a tumor suppressor, it was found lower expression in NSCLC tissues and cell lines. Moreover, SLC7A2 could increase drug sensitivity, immune infiltration and survival in NSCLC.Additional file 1: Figure S1A The heatmap analyses of the DEGs about SLC superfamily members between A549-WT and A549-WT cells treated with metformin. B The relative SLC7A2 mRNA expression in A. Figure S2 The 421 higher SLC7A2 and 92 lower SLC7A2 expressed LUAD patients’ prognostic information of Fig. p = 0.092. Table S1 The correlation between SLC7A2 expression and clinical parameters in NSCLC patients


Animal models of spontaneous osteoarthritis (OA) are sparse and not well characterized. The purpose of the present study is to examine OA-related changes and mechanisms in senescence-accelerated mouse prone 8 (SAMP8) that displays a phenotype of accelerated aging. Knees of male SAMP8 and SAM-resistant 1 (SAMR1) mice as control from 6 to 33 weeks of age were evaluated by histological grading systems for joint tissues , and µCT analysis. Gene expression patterns in articular cartilage were analyzed by real-time PCR. Immunohistochemistry was performed for OA-related factors, senescence markers, and apoptosis.Starting at 14 weeks of age, SAMP8 exhibited mild OA-like changes such as proteoglycan loss and cartilage fibrillation. From 18 to 33 weeks of age, SAMP8 progressed to partial or full-thickness defects with exposure of subchondral bone on the medial tibia and exhibited synovitis. Histological scoring indicated significantly more severe OA in SAMP8 compared with SAMR1 from 14 weeks [median (interquartile range): SAMR1: 0.89 (0.56–1.81) vs SAMP8: 1.78 (1.35–4.62)] to 33 weeks of age [SAMR1: 1.67 (1.61–1.04) vs SAMP8: 13.03 (12.26–13.57)]. Subchondral bone sclerosis in the medial tibia, bone mineral density (BMD) loss of femoral metaphysis, and meniscus degeneration occurred much earlier than the onset of cartilage degeneration in SAMP8 at 14 weeks of age.SAMP8 are a spontaneous OA model that is useful for investigating the pathogenesis of primary OA and evaluating therapeutic interventions.The online version contains supplementary material available at 10.1186/s13075-022-02916-5. Osteoarthritis (OA), the most common joint disease, is characterized by joint pain and decreased joint function and caused by various risk factors such as aging, traumatic injury, and obesity. The disease process is associated with changes in all joint tissues, including cartilage, subchondral bone, trabecular bone, synovium, menisci, and ligaments . The mecSpontaneous OA in mice develops much more slowly than in induced OA models. In C57BL/6 J OA-prone mice, OA-like changes only become detectable at 12–18 months –6. Thus The purpose of the present study is to examine OA-related changes and mechanisms in SAMP8 that displays a phenotype of accelerated aging.n = 10; SAMP8 n = 10 at 6, 9, 11, 14, 18, 28, and 33 weeks of age; SAMR1 n = 20; SAMP8 n = 20 at 23 weeks of age). Furthermore, SAMR1 and SAMP8 at 4 weeks of age (n = 15 for each strain) were used for in vitro studies. All mice were housed in groups of three to five per cage (S 143 mm × 293 mm × H148mm) with a sterilized beta-chip bedding and maintained at 23 ± 1 °C with a 12-h light/dark cycle and acidified water and complete commercial pelleted food ad libitum. All animal experiments were performed according to protocols approved by the Hiroshima University Animal Care and Use Committee.SAMP8 and SAMR1 at 3 weeks of age were obtained from Japan SLC , and only male mice were used in this study as only male mice are commercially available in Japan. A total of 115 SAMR1 and 115 SAMP8 were obtained from the vendor. For the aging study, a total of 90 mice each for SAMR1 and SAMP8 were evaluated in histopathological assessments  for 48 h at 4 °C and decalcification in K-CX  for 6 h at room temperature. Paraffin-embedded knee joints were sectioned at 4.5 μm in the coronal plane through the central weight-bearing region of the anterior and posterior femorotibial joint. Hip and ankle joints were sectioned in the sagittal plane. Three different sections per joint were stained with Safranin O  and Fast Green . Histological assessments were performed on each section, and the average scores from three different sections were used for statistical analysis. Three different researchers were blinded while performing all scorings. In this study, we applied separate scoring systems for articular cartilage, meniscus, subchondral bone, and synovium. Damage of articular cartilage  was evaluated using the OARSI scoring system . The criMouse lower limb was collected from SAMR1 and SAMP8 at 4, 6, 9, and 23 weeks of age and was kept in 70% EtOH after fixation in 4% paraformaldehyde phosphate buffer solution (PBS) for 48 h at 4 °C. Analysis by µCT was performed in the epiphysis of the tibias and the metaphysis of the distal femur as we previously described .n = 4) and SAMP8 (n = 4) at 9 weeks of age. Measurement of IL-6 in serum was carried out using Milliplex MAP technology. Multiplex Assay kit  was obtained, and the procedure was performed following the manufacturer’s instruction.Mouse serum was collected from abdominal vena cava in SAMR1 , anti-ADAMTS-5 antibody , and anti-MMP13 antibody  diluted in Can Get Signal immunostaining solution  using Vectastain ABC-AP alkaline phosphatase kit and AP substrate kit  according to the manufacturers’ instructions. For type X Collagen staining, slides were pretreated with antigen-retrieval reagent  at room temperature for 10 min, blocking serum for 30 min. Then, sections were immunostained with anti-type X Collagen antibody  diluted in PBS using VECTASTAIN Elite ABC-HRP kit and DAB substrate kit. Mouse IgG1 kappa , mouse IgG2b kappa (14–4731-82), and rabbit IgG (14–4616-82) were used as isotype control antibodies for negative controls  at 60 °C for 16 h, followed by blocking with 10% normal horse serum for 30 min. Then, sections were immunostained with anti-P16TUNEL staining was completed using an in situ detection kit for programmed cell death detection  according to the manufacturer’s instructions. Nuclei were stained by 4’,6-diamidino-2-phenylindole (DAPI).Articular cartilage tissues for gene expression analysis were isolated from knee joints of mice at 4 weeks of age under a microscope. For chondrocyte isolation, cartilage was resected from the femoral heads of SAMR1 and SAMP8 at 4 weeks of age and digested with 3.5 mg/ml collagenase Type 2  in Dulbecco’s modified Eagle’s medium (DMEM)  for 1.5 h at 37 °C. Primary chondrocytes were seeded on a 12-well plate with DMEM with 10% fetal bovine serum and 1% penicillin/streptomycin and cultured for 7–10 days. Then, the chondrocytes (passage 1) with or without IL-1β  were used in the present experiments.Gapdh was used as the internal control to normalize the sample differences. Relative expression was calculated using the ΔΔCt values, and results were expressed as 2−ΔΔCt.Total RNA was extracted from articular cartilage tissues and cultured chondrocytes using Isogen reagent  and RNA purification kit . Complementary DNA (cDNA) was synthesized with a Reverse Transcription system  according to the manufacturer’s protocol. Quantitative polymerase chain reaction (PCR) was performed with the TaqMan Gene Expression Assay probes Table S  were harvested from 4-week-old SAMR1 and SAMP8 mice and weighed. The amount of the released glycosaminoglycan into the conditioned medium was measured using the Blyscan Glycosaminoglycan assay kit  as previously described . After nn = 5) and the SAMP8 group (n = 5) for checking sample size showed an effect size of 2.5 by power analysis using G*power (version 3.1.9.2) . Using this effect size, we calculated the sample size (the power of 0.8 and a significance level of 0.05) and found that at least 6 animals were required in each group. To account for potential dropouts and increase power, the number of animals was increased to 10 in each group (only 23-week-old mice are n = 20 in each group). Thus, we evaluated SAMR1 and SAMP8 as follows SAMR1 n = 10; SAMP8 n = 10 at 6, 9, 11, 14, 18, 28, and 33 weeks of age. For the 23-week time point, we allocated 20 mice per strain to establish as reasonable point for OA evaluation because SAMP8 reliably exhibited OA-like pathologic changes in all joint tissues . Actual measurement value data are presented as mean ± standard deviation (S.D). Comparison of mean values was performed using Welch’s t test. The p-values were corrected using the Holm-Sidak method for multiple comparison (GraphPad Prism9). Data of body weight was analyzed by two-way factorial ANOVA. Scoring data were compared between SAMR1 vs SAMP8 in aging analysis with Mann–Whitney U test at each time point. Comparison of right and left knees of SAMP8 mice were performed using the Wilcoxon signed-rank test at each time point. The p-values were corrected by using Holm’s method for multiple comparison correction . Kendall’s tau-b partial correlation coefficient was used to examine the correlation between the data of OARSI, meniscus, synovial, and subchondral bone scoring. Differences were considered statistically significant at # = P < 0.05 and ## = P < 0.01.In a pilot study, our comparison of the 23-week-old OARSI score between the 23-week-old SAMR1 group  at 9 weeks of age Fig. A. SubchoTo examine inflammatory condition in SAMP8, inflammatory cytokine in serum was measured. However, IL-6 levels were low in both SAMR1 and SAMP8  at 9 weeks of age before the onset of cartilage damage (data not shown).We focused on the events in articular cartilage that occur at 4 to 11 weeks of age before the onset of macroscopic and histological cartilage degradation. Chondrocyte hypertrophy in articular cartilage has been characterized as a pathological change of OA and inhibiting chondrocyte hypertrophy has been considered as a target for OA treatment –20. The INK4a, a marker of senescent or dysfunctional chondrocytes [INK4a-positive senescent cells were detected, SAMP8 did not exhibit severe OA in hip and ankle joints at 23 weeks  factor IL-6 were undetected in articular cartilage. Cultured articular chondrocytes showed that basal levels or IL-1β induced changes of Adamts5 was significantly increased in SAMP8, while Col2a1, Acan, and Mmp13 were not different between SAMR1 and SAMP8 , meniscus degeneration and cartilage damage in medial compartment , and subchondral bone sclerosis and cartilage damage . These results indicated that the cartilage damage in SAMP8 is associated with the pathologic changes of knee joint tissues as typical OA pathology.Finally, we analyzed the correlation of pathological changes between joint tissues  of 14–33-week-old SAMP8 mice Table S. There wOA, an aging-related disease, develops slowly over a long period of time. Thus, in most mouse strains, OA-like structural changes with severe cartilage damage are slow to progress and are not detectable until around 15–22 months of age –6. In adAlthough the reasons why SAMP8 display accelerated aging and various disease are still unknown, different combinations of mutations in disease-causing genes may be responsible for the various phenotypes of SAMP strains . SAMP8 dINK4a-positive senescent cells increase in articular cartilage with aging, but p16INK4a-positive cells are not essential for SASP production and are not involved in the development of OA [INK4a is recognized as senescent cell marker, relation between accelerated-aging phenotype and p16INK4a-positive senescent cells in SAMP8 has not been characterized yet. In the present study, although many chondrocytes in SAMP8 at 4 weeks of age did not express the senescence marker p16INK4a, the rate of p16INK4a-positive chondrocytes in SAMP8 was increased with aging than in SAMR1. However, p16INK4a was also detected in chondrocytes of the lateral tibial plateau, ankle, and hip joint of SAMP8 which showed less cartilage degeneration than the medial compartment of knee joints at 23 weeks of age. Furthermore, ankle and hip joints expressed p16INK4a but did not exhibit OA-like cartilage defects even at 52 weeks of age (Fig. SINK4a-positive chondrocytes in the articular cartilage might not be an essential trigger in the development and progression of OA in SAMP8.Chondrocyte hypertrophy and senescent cells increased at 6 weeks before the onset of cartilage degradation at 14 weeks of age. Increased numbers of hypertrophic chondrocytes in articular cartilage have been observed in OA development, and inhibition of hypertrophy has been shown to reduce the severity of OA –20. Age-nt of OA . AlthougINK4a [OA is whole joint disease , and lesINK4a . AlthougINK4a , adiposiINK4a , 46. HowINK4a . Thus, iINK4a . AlthougINK4a . The relINK4a , althougTogether, our study indicates that SAMP8 is a spontaneous and early-onset OA mouse model that is useful for evaluating OA pathogenesis. SAMP8 exhibited severe OA with the pathologic changes of knee joint tissues. A better understanding of the genes, molecules, and processes involved in the OA pathogenesis of SAMP8 will therefore contribute significantly to the identification of new preventative, protective, and therapeutic approaches for primary OA.Additional file 1:Fig. S1. Incidence of aging-related spontaneous osteoarthritis development in knees of SAMP8 at 23 weeks of age.Additional file 2:Fig. S2. Histology of whole knee joint in SAMR1 and SAMP8.Additional file 3:Fig. S3. Hip and ankle joints of SAMP8 with aging.Additional file 4:Fig. S4. ADAMTS-5 and MMP-13-positive in medial and lateral compartment of the knee from SAMR1 and SAMP8.Additional file 5:Fig. S5. Isotype control Images of all Antibodies used in Immunohistochemistry.Additional file 6: Supplementary materials and methods.Additional file 7:Table S1. OARSI score.Additional file 8:Table S2. Meniscus score.Additional file 9:Table S3. Synovitis score.Additional file 10:Table S4. Subchondral bone score.Additional file 11: Table S5.Additional file 12: Table S6.Additional file 13: Table S7.