The nanoparticles decreased the initial rate of ʟ-[14C]glutamate uptake, and increased the ambient level of ʟ-[14C]glutamate in isolated cortex nerve terminals (synaptosomes). The nanoparticles exhibit a high capability to adsorb glutamate/ʟ-[14C]glutamate in water. Some components of the incubation medium of nerve terminals, that is, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and NaH2PO4, decreased the ability of γ-Fe2O3 nanoparticles to form a glutamate biocoating by about 50% and 90%, respectively. Only 15% of the amount of glutamate biocoating obtained in water was obtained in blood plasma. Albumin did not prevent the formation of a glutamate biocoating. It was shown that the glutamate biocoating is a temporal dynamic structure at the surface of γ-Fe2O3 nanoparticles. Also, components of the nerve terminal incubation medium and physiological fluids responsible for the desorption of glutamate were identified. Glutamate-coated γ-Fe2O3 nanoparticles can be used for glutamate delivery to the nervous system or for glutamate adsorption (but with lower effectiveness) in stroke, brain trauma, epilepsy, and cancer treatment following by its subsequent removal using a magnetic field. γ-Fe2O3 nanoparticles with transient glutamate biocoating can be useful for multifunctional theranostics.Glutamate is the main excitatory neurotransmitter in the central nervous system and excessive extracellular glutamate concentration is a characteristic feature of stroke, brain trauma, and epilepsy. Also, glutamate is a potential tumor growth factor. Using radiolabeled ʟ-[ The transporters use Na+/K+ electrochemical gradients across the plasma membrane to accomplish glutamate transport.Glutamate is a main fast excitatory neurotransmitter in the central nervous system. Normal brain function in a majority of its aspects involves glutamate signaling. The extracellular glutamate level is low between episodes of exocytotic release under normal physiological conditions, thereby preventing continual activation of glutamate receptors and protecting neurons from excitotoxic injury . Each syGlutamate is a potential growth factor in tumor development. The extracellular glutamate concentrations in glioma cell lines in vitro was shown to be up to 500 µM, and glutamate stimulates glioma cell proliferation in vivo. Also, glial tumor cells ex vivo generate neurotoxic quantities of glutamate –6. Exces2O3 nanoparticles are very promising in targeted drug delivery, cancer therapy, diagnostics, and hyperthermia treatment due to their magnetism and chemical stability glutamate and scintillation counting; (ii) to analyze the adsorption of glutamate on γ-Fe2O3 nanoparticles forming a biocoating and, vice versa, the loss of the glutamate biocoating in a standard salt solution and physiological fluids; and (iii) to assess the effects of the nanoparticles on key characteristics of glutamatergic neurotransmission in rat brain terminals (synaptosomes). Glutamate biocoating of γ-Fe2O3 nanoparticles for glutamate delivery to and removal from the nerve terminals and other cells can be very promising in the treatment of stroke (damaged core and penumbra zones), brain trauma, epilepsy, and cancer.The aims of this study were: (i) to develop new approach of glutamate adsorption measurements in different media using radiolabeled ʟ-glutamate . The synaptosomal suspension was further incubated at 37 °C for 1 min, and then sedimented using an Eppendorf microcentrifuge at 10,000g for 20 s. The ʟ-[14C]glutamate uptake was calculated as the decrease of radioactivity in supernatant aliquots of 100 μL and the increase of radioactivity in the pellets (preliminary treated with sodium dodecyl sulfate) using liquid scintillation counting with ACS scintillation cocktail (1.0 mL) using a Delta 300 Tracor Analytic scintillation counter (USA).The uptake of ʟ-glutamate  at 37 °C for 10 min. After loading, the synaptosomal suspensions were washed and the pellets were resuspended again in the standard salt solution to a final concentration of 1 mg protein per mL and immediately used in the experiments. Monitoring the extracellular ʟ-[14C]glutamate level was accomplished as follows: The synaptosomal suspensions  were preincubated at 37 °C for 10 min. Then, γ-Fe2O3 nanoparticles were added and further incubated for 0 min and 6 min and then sedimented using an Eppendorf microcentrifuge at 10,000g for 20 s. Radioactivity was measured in supernatant aliquots of 100 µL using liquid scintillation counting and was expressed as a percentage of the total ʟ-[14C]glutamate incorporated into the nerve terminals glutamate  and non-radiolabeled glutamate at definite concentrations were mixed with abovementioned components along with different amounts of γ-Fe2O3 nanoparticles also added to the probe. The incubation time was 5 min in the salt solutions and 30 min in blood plasma or albumin. After that a magnetic field was applied or the nanoparticles were sedimented in a microcentrifuge . Adsorption was measured in the aliquots of the supernatants (100 µL) by liquid scintillation counting with ACS scintillation cocktail (1.5 mL), expressed as percentage of the total amount of radiolabelled glutamate adsorbed. The ʟ-[14C]glutamate count at definite concentrations was considered as 100% in further calculations.Glutamate adsorption by γ-Fe2O3 nanoparticles was measured by dynamic light scattering using a laser correlation spectrometer ZetaSizer-3  equipped with a He–Ne laser LGN-111 . A suspension of γ-Fe2O3 nanoparticles (1 mL) in the standard salt solution was placed to a cylindrical quartz cuvette of 10 mm in diameter, which was inserted into the laser correlation spectrometer. The measurement range of the instrument is from 1 nm to 50 µm. Registration and statistical processing of laser light scattered by a water (n = 1.33) suspension of the nanoparticles, measured repeatedly for 120 s at 22 °С at a scattering angle of 90°, were carried out. The results of the experiments were processed using the software PCS-Size mode v1.61. The laser correlation spectrometer was equipped with a multi-computing correlator type 7032 CE.The size of γ-Fe2O3 nanoparticles were performed using software for graphic-mathematical modeling, that is, a demonstration version of Diamond 4.5.3  for the simulation of nanoparticle polycrystals, a demonstration version of LeadIT 2.3.2 (BioSolveIT GmbH©) to find the most probable binding sites between protein and nanoparticles, a free version of ArgusLab 4.0.1 (Mark Thompson and Planaria Software LLC©) for modeling a biomodified nanoparticle, and a trial version of the Materials Sciences Suite 2015 (Schrödinger Software©) software package to generate appropriate images of the whole protein–nanoparticle cluster.Simulations of biocorona formation at the surface of γ-Fen independent experiments. One-way ANOVA followed by post hoc Tukey’s test was used for data processing. The accepted level of significance was set as P < 0.05.The results were expressed as mean ± S.E.M. of 2O3 were prepared by the coprecipitation method and oxidation with sodium hypochlorite. According to TEM  for 5 min in water or salt solutions and for 30 min in plasma or albumin. To avoid any contribution of unbound glutamate/ʟ-[14C]glutamate, the γ-Fe2O3 nanoparticles were isolated from the incubation medium using a magnetic field . Alternatively, the nanoparticles were sedimented in a microcentrifuge . After removal of the nanoparticles from the incubation media, they were washed repeatedly with 10 volume equivalents of water. The formation of glutamate biocoating was measured as a decrease in radioactivity in the aliquots of the incubation medium (100 mL) by liquid scintillation counting with ACS scintillation cocktail (1 mL). The value of ʟ-[14C]glutamate radioactivity at definite concentrations in the corresponding incubation medium with no nanoparticles added was used in calculations as a control (100%) and consisted of 30,000 cpm/mL at 1.86 µM glutamate/ʟ-[14C]glutamate. The results were expressed as percentage of the total amount of ʟ-[14C]glutamate in the control.Using radiolabeled ʟ-glutamate count at the corresponding concentrations in each tetrad was considered as 100% in further calculations  = 21.6, Р < 0.001, n = 5)  = 92.2, Р < 0.001, n = 5). Nanoparticles at a concentration of 0.75 mg/mL were able to adsorb 37.7% ± 2.0% of 20 µM glutamate/ʟ-[14C]glutamate, at a concentration of 1.5 mg/mL they adsorbed 61.7% ± 1.7% of 20 µM glutamate/ʟ-[14C]glutamate, and at a concentration of 3 mg/mL they adsorbed 84.5% ± 1.3% of 20 µM glutamate/ʟ-[14C]glutamate  = 332.6, Р < 0.001, n = 5).The first sets of the experiments were aimed to assess the glutamate biocoating of γ-Feefficacy –27. The ulations . It was , n = 5) . Nanopar14C]glutamate uptake by isolated nerve terminals (synaptosomes). The latter is an excellent system to analyze presynaptic transport processes [2O3 nanoparticles at a concentration of 1 mg/mL decreased the initial rate of synaptosomal ʟ-[14C]glutamate uptake by approximately 25%  = 6.5 , Р < 0.05, n = 4).The capability of nanoparticles to form a glutamate biocoating shown in the previous subsection can change the effectiveness of ʟ-4Cglutamately 25% . The ini14C]glutamate adsorption by γ-Fe2O3 nanoparticles, it is logical that the nanoparticles can decrease also the ambient ʟ-[14C]glutamate concentration in the synaptosomal preparations. However, γ-Fe2O3 nanoparticles increased the synaptosomal ambient level of ʟ-[14C]glutamate  = 7.5, Р < 0.05, n = 4). When the concentration of γ-Fe2O3 nanoparticles was decreased to 50 and 1 µg/mL, no significant changes were revealed in the ambient level of ʟ-[14C]glutamate in nerve terminal preparations. The levels were 0.195 ± 0.017 and 0.210 ± 0.019 nmol/mg of protein, respectively. Hence, only at a high concentration of 1 mg/mL did the nanoparticles influence the initial rate of ʟ-[14C]glutamate uptake and the ambient level of ʟ-[14C]glutamate in the nerve terminal preparations.If a decrease in the uptake was a result of ʟ-glutamate cannot be used because of poor probe transparency. Hence, in this set of the experiments lower concentrations of γ-Fe2O3 nanoparticles were used. It was demonstrated that γ-Fe2O3 nanoparticles at low concentrations did not influence the fluorescence intensity of rhodamine 6G in the synaptosomal preparations, which in turn reflected an absence of membrane depolarization in the nerve terminals.The plasma membrane potential  surface species and a carboxylate group glutamate biocoating at the nanoparticle surface. As shown in 2O3 nanoparticles, at concentrations of 0.75, 1.5, and 3 mg/mL, to adsorb 1.86 µM glutamate/ʟ-[14C]glutamate compared to an aqueous solution. Also, an almost complete inhibition of glutamate/ʟ-[14C]glutamate biocoating at the nanoparticle surface was shown with 1 mM NaH2PO4. In particular, at a nanoparticle concentration of 0.75 mg/mL, the adsorption of 1.86 µM glutamate/ʟ-[14C]glutamate was 83.0% ± 1.7% in 10 mM KCl, 53.0% ± 1.6% in 20 mM HEPES, and 1.6% ± 0.3% in 1 mM NaH2PO4  = 943.4, Р < 0.001, n = 4), respectively. At a nanoparticle concentration of 1.5 mg/mL, the adsorption of 1.86 µM glutamate/ʟ-[14C]glutamate was 91.0% ± 2.0% in 10 mM KCl, 74.0% ± 2.1% in 20 mM HEPES, and 2.5% ± 0.5% in 1 mM NaH2PO4  = 761.4, Р < 0.001, n = 4), respectively. At a nanoparticle concentration of 3 mg/mL, the adsorption of 1.86 µM glutamate/ʟ-[14C]glutamate was 93.0% ± 1.0% in 10 mM KCl, 83.0% ± 1.9% in 20 mM HEPES, and 1.1% ± 0.2% in 1 mM NaH2PO4  = 1544.3, Р < 0.001, n = 4).The components of the standard salt solution  revealed that the overall mean was 171 nm in water , which is 564 nm . HEPES asorption .14C]glutamate biocoating at the surface of γ-Fe2O3 was monitored in blood plasma. It was revealed that blood plasma significantly decreased the adsorption ability of the nanoparticles. The nanoparticles at concentrations of 0.75, 1.5 and 3 mg/mL adsorbed 3.3% ± 0.5%, 6.7% ± 1.5% and 14.7% ± 1.4% of 1.86 µM glutamate/ʟ-[14C]glutamate, respectively, in the presence of blood plasma  = 23.1, Р < 0.001, n = 4)  = 1.0, Р > 0.05, n = 4). Therefore, not albumin, but other components of blood plasma caused a decrease in the glutamate biocoating capability of γ-Fe2O3 nanoparticles.In this set of the experiments, the glutamate/ʟ-.–32.28–322O3 nanoparticle surface glutamate and physiologically compatible media. The possibility of modulating the glutamate transport in nerve terminals and blood plasma was analyzed. It was revealed that 90% of glutamate/ʟ-[14C]glutamate was bound to the nanoparticles in water  receptors [2O3 nanoparticles can more easily penetrate the blood–brain barrier than uncoated ones. Glutamate adsorbed on γ-Fe2O3 nanoparticles in water is expected to be partially released from the nanoparticle surface after contact with biological fluids. Application of magnetic fields can make this a targeted process.Effective magnetic field-mediated glutamate delivery using γ-Fenslation . It was nslation . The neueceptors . Glutamaeceptors . Thus, i2O3 nanoparticles can be used (but with lower effectiveness) for glutamate adsorption in the organism followed by its consequent removal using magnetic fields. Glutamate adsorption at the nanoparticle surface and its removal efficiency in blood plasma is only 15% of that in water. It is well-established that high extracellular glutamate levels in the nerve cells lead to excitotoxicity and cell death in stroke and brain trauma. Epilepsy seizures are also accompanied with increased extracellular glutamate concentrations. The balance between the interstitial glutamate concentration in the brain and in the blood was established [2O3 nanoparticles to adsorb glutamate can be used for the prevention of neurotoxicity in the blood and also in the brain and peripheral nervous system after further technological optimization.Vice versa, γ-Feablished . Also, aablished . In this2O3 nanoparticles not only from brain cancer but also from peripheral tissue cancer. It was shown that cells from non-CNS cancers may secrete glutamate into their extracellular environment [62O3 nanoparticles can be potentially useful in stroke, brain trauma, epilepsy, and cancer treatment.In addition, glutamate is a potential growth factor in tumor development. Glutamate can be removed using γ-Feironment –643–45. 2O3 nanoparticles. Compounds of the synaptosomal incubation medium that caused a removal of the glutamate biocoating were identified. It was found that blood plasma components other than albumin are responsible for glutamate desorption from the surface of γ-Fe2O3 nanoparticles. Glutamate-conjugated γ-Fe2O3 nanoparticles can be used for neurotransmitter delivery for modulation of extracellular glutamate homeostasis and synaptic neurotransmission. Vice versa, these nanoparticles can be used (but with lower efficacy) for glutamate adsorption in different tissues following by its consequent removal with magnetic fields.This study demonstrated that glutamate biocoating is a temporal dynamic structure at the surface of γ-FeFile 1Simulation of the spatial structure of a maghemite nanoparticle coated with a blood plasma protein biocorona.


The complement system has developed different strategies to clear infections by several effector mechanisms, such as opsonization, which supports phagocytosis, attracting immune cells by C3 and C5 cleavage products, or direct killing of pathogens by the formation of the membrane attack complex (MAC). As the Zika virus (ZIKV) activates the classical complement pathway and thus has to avoid clearance by the complement system, we analyzed putative viral escape mechanisms, which limit virolysis. We identified binding of the recombinant viral envelope E protein to components of the terminal pathway complement  by ELISA. Western blot analyses revealed that ZIKV E protein interfered with the polymerization of C9, induced on cellular surfaces, either by purified terminal complement proteins or by normal human serum (NHS) as a source of the complement. Further, the hemolytic activity of NHS was significantly reduced in the presence of the recombinant E protein or entire viral particles. This data indicates that ZIKV reduces MAC formation and complement-mediated lysis by binding terminal complement proteins to the viral E protein. The complement system is an effective arm of innate immunity. It is a family of membrane-anchored and soluble proteins circulating in the blood in their inactive form , 2. Uponvia endocytosis (receptor-mediated endocytosis) , yellow fever (JFV), West Nile (WNV), Japanese encephalitis (JEV), Zika virus (ZIKV), and the closely related hepatitis C virus (HCV), all of which share close similarities in structure , 13. Thecytosis) . NS protcytosis) , 10. In cytosis) , 17. Furcytosis) . Althougcytosis) , 19.Our data indicates that, similar to NS1, the E protein binds to terminal pathway complement proteins, interferes with the formation of MAC on the surface of the cells and further reduces complement-mediated lysis.Purified complement proteins C5b6 (MW 285 kDa), C7 (MW 91.4 kDa), C8 (MW 151 kDa), and C9 (MW 71 kDa) were purchased from Complement Technology . ZIKV recombinant E and NS1 proteins were provided by Aviva Systems Biology ; E protein PMA04848-1MG and Biozol ; E protein MBS596001, and Zika NS1 MBS596002). Anti-flavivirus group antigen antibody [D1-4G2-4-15 (4G2)] and goat anti-mouse antibody conjugated to horseradish peroxidase were purchased from Szabo-Scandic .Recombinant human vitronectin was obtained from BioLegend . Bovine serum albumin (BSA) was provided by Carl-Roth .Anti-C9  was purchased from Hycult . Normal human serum (NHS) was acquired from Dunn Labortechnik GmbH  and stored in aliquots at −80°C. Anti-human HLA-ABC antigen clone W6/32 was obtained from Agilent Technologies Dako . C9 depleted serum was generated as described in detail .A549 cells for Western blot and virus production and Aedes albopictus C6/36 mosquito cells for virus propagation were kindly provided by Prof. Dr. Karin Stiasny, Medical University of Vienna. Sheep erythrocytes for the hemolysis assay were obtained from Virion . Two strains of the virus, MRS_OPY_Martinique_PaRi_2015 (GenBank: KU647676) and ZIKV strain MR766 (GenBank: DQ859059) were kindly provided by the European Virus Archive . The virus was propagated as described elsewhere .Cell lysates were analyzed by western blot, RIPA buffer for inducing lysis of the A549 cells was purchased from Cell Signaling Technology . Veronal-buffered saline (VBS) was provided by Virion. Dulbecco’s modified Eagle’s medium (DMEM) and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich .Complement proteins including C5b6, C7, C8, and C9 in 1:2 dilutions starting from 5 µg/ml were coated on the microtiter 96 well ELISA plate and incubated overnight at 4°C. After washing with PBS-Tween 0.01%, E protein (10 µg/ml) was added to each well and incubated for 1 h at room temperature (RT) with slow continuous shaking. The ELISA plate was washed three times and blocked with 5% BSA for 1 h. Antibody against envelope protein (4G2) was added at a concentration of 1:500 and incubated for 1 h. Finally, horseradish peroxidase-labeled goat anti-mouse antibody was added . The TMB substrate from Sera Care  was used. The optical density (OD) was measured at a wavelength of 650 nm. To test if native C9 from NHS was also able to bind to the E protein, serial dilutions of NHS were incubated with a constant amount of E-protein (10 µg/ml coated overnight in the ELISA plate. BSA at the same concentration (10 µg/ml) or dilutions of C9-depleted serum (ΔC9 NHS) were applied as negative controls. Anti-C9 (WU 13-15) at a concentration of 1 µg per well was added. After washing, samples were incubated with secondary antibody  and finally, TMB substrate solution. Optical density (OD) was measured at a wavelength of 650 nm.2 at 37°C. In parallel, the E protein was incubated with 5% NHS at 37°C for 30 min a total volume of 300 µl for each reaction). After three washing steps of the cells with VBS, the mixture of the E protein and NHS was added to the cells and incubated for 60 min at 37°C. Cells were washed again, lysed on ice with RIPA buffer for 30 min (100 µl of lysis buffer), and the lysate was loaded on an 8% acrylamide gel under non-reducing conditions. Lysates were blotted and the membrane was blocked with 5% nonfat dried milk in Tris-buffered saline with 0.1% Tween20 (TBST) for 60 min. The first antibody against C9 protein (WU 13-15)  was added to the blocking solution and incubated overnight at 4°C. The following day, the blot was washed three times with TBST and a horseradish peroxidase-labeled goat anti-mouse antibody was added . After incubation for 2 h at room temperature, the membrane was washed three times and developed using the ImageQuant LAS-4000 . In further assays, anti-human HLA-ABC was used in a sublytic amount  as an activator of the classical pathway (instead of purified C5b6 protein). Ab was added to the A549 cells and incubated for 60 min at 37°C. In parallel, different amounts of E protein were incubated with 5% NHS at 37°C for 30 min. After washing the cells with VBS, the mixture of the E protein and NHS was added to the cells and incubated for 50 min at 37°C. Deposition of C9 on the cell surface was analyzed by western blotting as described above.A C9 polymerization assay was performed to study the formation of a membrane attack complex in the presence or absence of E protein. For this, A549 cells were seeded a day before the experiment at 1 × 10exp5 cells per well in 24 well plates purchased from Szabo-Scandic  in complete DMEM , 2 mM L-glutamine, 100 units/mL penicillin G, 100 µg/ml streptomycin). The next day, cells were washed three times with VBS and C5b6 protein (5 µg in 300 µl) was added to the cells and incubated for 2 h in a humidified incubator supplied with 5% CO8 cells/ml) resuspended in VBS were sensitized with C5b6 (1 µg) for 60 min at RT using a U-bottom microtiter plate from Greiner Bio-one . In a separate preparation, E protein (10 µg), Vn (20 µg), and mixtures of Vn-and NS1 (containing 20 µg VN and 10 µg NS1) and Vn and-E protein (containing 10 µg E protein and 20 µg Vn) were each incubated with C7-C9 (C7 (1 µg), C8 (0.5 µg), and C9 (1 µg) for 15 min at 37°C. Next, the prepared mixtures were added to the sheep erythrocytes coated with C5b6 and incubated for 30 min at 37°C in a total volume of 100 µl per reaction (8 cells/ml) were added to the samples and the mixture was incubated for 30 min at 37°C. The amount of hemoglobin released from the lysed cells was measured by determining the absorbance of the supernatant at an optical density (OD) of 415 nm. To distinguish between the effects of NS1 or E-Protein on the reduction of hemolysis, viral proteins were incubated separately or as a mixture with NHS (1:160 in VBS) before sensitized sheep erythrocytes were added and the lysis assay was performed as described above.To analyze the activity of the complement system, sheep erythrocytes  was considered statistically significant .As ZIKV activates the classical pathway of complement, we were interested in whether the virus adapted means to reduce virolysis. In a first attempt, we assessed whether purified C7, C8, or C9 bind ZIKV E in ELISAs. In contrast to C7, both C8 and C9 interacted with the viral recombinant E protein , lysis induced by E protein was not further enhanced when both proteins were co-applied. In contrast, hemolysis was enhanced by vitronectin when the protein was added together with NS1, which confirms the recently published data of Conde et al. . As we wIn summary, not only NS1, but also ZIKV E protein can reduce the formation of the MAC. As ZIKV activates the classical pathway by direct binding of C1q to the E protein and infection by this virus upregulates the expression of complement proteins , the virThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.ZM, BS, and SB: experimental work. ZB, RW, and HS: study design, data interpretation, drafting the article, critical revision of the article, and final approval. All authors contributed to the article and approved the submitted version.This study was supported by the FWF, Vienna Austria .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arabidopsis thaliana SCD1  that encodes a RAB-guanine nucleotide exchange factor. Auxin transport, lateral root initiation, asymmetric auxin-induced gene expression after gravitropic reorientation, and differential gravitropic growth were reduced in the roots of the scd1-1 mutant relative to wild type at the restrictive temperature of 25°C, but not at the permissive temperature of 18°C. In scd1-1 at 25°C, PIN1- and PIN2-GFP accumulated in endomembrane bodies. Transition of seedlings from 18 to 25°C for as little as 20 min resulted in the accumulation of PIN2-GFP in endomembranes, while gravitropism and root developmental defects were not detected until hours after transition to the non-permissive temperature. The endomembrane compartments that accumulated PIN2-GFP in scd1-1 exhibited FM4-64 signal colocalized with ARA7 and ARA6 fluorescent marker proteins, consistent with PIN2 accumulation in the late or multivesicular endosome. These experiments illustrate the power of using a temperature sensitive mutation in the gene encoding SCD1 to study the trafficking of PIN2 between the endosome and the plasma membrane. Using the conditional feature of this mutation, we show that altered trafficking of PIN2 precedes altered auxin transport and defects in gravitropism and lateral root development in this mutant upon transition to the restrictive temperature.Auxin is transported in plants with distinct polarity, defined by transport proteins of the PIN-formed (PIN) family. Components of the complex trafficking machinery responsible for polar PIN protein localization have been identified by genetic approaches, but severe developmental phenotypes of trafficking mutants complicate dissection of this pathway. We utilized a temperature sensitive allele of  Arabidopsis roots, PIN1 is localized to the stele and implicated in auxin transport toward the root apex ], at 18°C and at 25°C. At 18°C, Col(g) and scd1-1 had similar primary root elongation rates and lateral root numbers  (scd1-1 was significantly impaired relative to Col(g) at both temperatures with p < 0.01 for all comparisons.If  numbers . At 25°Cn Col(g) , and then Col(g) . We perfscd1-1 mutant allowed us to ask if shorter-term growth at 25°C could induce this lateral root phenotype and whether the impaired root development at this restrictive temperature could be rescued by transition back to the permissive temperature. Col(g) and scd1-1 seedlings were grown at constant 18°C, or constant 25°C, or grown at 18°C and then transferred to 25°C after 2, 4, or 6 days  and decreased lateral root formation in scd1-1. When scd1-1 seedlings were grown at 18°C for 2 days before transfer to 25°C, significantly fewer lateral roots formed than in plants grown continuously at 18°C, while roots grown at 18°C for 4 or 6 days before transfer to 25°C formed an equivalent number of lateral roots to the continuous 18°C scd1-1 control  and d 6 days  to deterscd1-1 seedling roots. Wild type and scd1-1 seedlings grown at 18 or 25°C for 5 days were rotated 90°  showed a more rapid response than at 18°C, with root curvature reaching 80° and 50° by 8 h at 25° and 18°C, respectively. In contrast, scd1-1 at 25°C showed a 50% reduction in the gravitropic response relative to Col(g), with root curvature reaching a mean angle of only 40°.We examined the gravity response in wild type and ated 90° . At 18°Cscd1-1 phenotype at the restrictive temperature on gravity response, we asked whether the gravity response would be impaired in plants grown at 18°C for 5 days and transferred to 25°C for 4–12 h prior to reorientation. The gravitropic response in the scd1-1 mutant 12 h after transition to 25°C was slower than Col(g), impaired to levels equivalent to seedlings grown continuously at 25°C (scd1-1 seedlings transferred from 18 to 25°C for 4 h before reorientation did not show a decrease in gravitropic curvature as compared to Col(g), while 8 h at 25°C led to a significant, but not complete, inhibition of curvature  and scd1-1 had similar numbers of statoliths per cell and number of columella cells, suggesting that the gravitropic defect in scd1-1 was not due to altered perception of the gravity vector  at 25°C. scd1-1 seedlings were grown at 18°C for 5 days then transferred to 25°C, and the amount of shootward 3H-IAA transport was determined. Within 8 h of transfer there was a significant decrease in IAA transport compared to wild type, with a further decrease by 24 h  were used to follow movement of an auxin-induced gene expression signal using a previously published method  were used to determine whether the altered gravitropic response of scd1-1 was accompanied by a temperature-sensitive defect in the lateral redistribution of auxin at the root tip. Seedlings were grown at 18°C for 5 days then were reoriented 90°, and then imaged at 2 h intervals after staining with propidium iodide (PI) to reveal the boundaries of cells. GFP fluorescence was captured in optical slices through the center of the root with settings optimized to visualize the signal in the epidermis on the lower side of the root. At 18°C roots of both genotypes showed asymmetric auxin-induced GFP fluorescence signal with the GFP signal moving a greater distance from the tip back on the lower side of roots beginning 4 h after reorientation  fusion protein was examined in wild type and scd1-1 seedlings  was also found in endomembrane bodies in scd1-1 mutants at 25°C, but not in wild type (PIN3pro::PIN3-GFP (PIN3-GFP) was not detected in endomembrane bodies. The localization of ABCB19 and AUX1 fused to fluorescent proteins was not altered in scd1-1  or PIN2-GFP fluorescence (green), with the majority of endomembrane bodies containing both fluorescent signals  and RFP-ARA7 (magenta) were grown at 25°C, endomembrane bodies were observed that have only PIN2-GFP signal (green) or RFP-ARA7 (magenta) signal, while a subset contain both signals (white)  . Higher ficients  for the ficients  were 0.8ficients . The preficients .scd1-1 accumulating in late multivesicular endosomes and not in other compartments.We found little or no overlap in the endomembrane localization of PIN2-GFP fluorescence in lines containing reporters localized in either the early endosome (VHA1-RFP)  TGN SYP, or the Polar transport of the hormone auxin from cell to cell is controlled by the asymmetric localization of the PIN family of IAA efflux carriers . These cscd1-1 mutant to uncover the dynamics of auxin transport targeting and how these reversible changes in protein targeting control auxin transport and dependent process. SCD1 contains a DENN domain found in proteins with RAB-directed GEF activity and in proteins that interact with clathrin ) showing reductions in the long distance rootward transport within 8 h after transition. A previous report suggested that there is specific regulation of auxin transport in this time window with a pulse of auxin moving from the shoot to the root at 3–5 days, which correlated with the emergence of the first lateral root  a transition from 18 to 25°C increases shootward transport by more than fivefold within 5 h, leading to a more than twofold greater transport in Col(g) than in scd1-1. Treatment of roots for 12 h at the non-permissive temperature impaired gravity response in scd1-1 to levels consistent with constitutive growth at this non-permissive temperature. Together these results support the timeline where PIN2 targeting is impaired, reducing auxin transport and then dependent physiological processes.The trafficking defect is not detectable in itropism . Upon trscd1-1. However, other fluorescent fusions to the auxin transport proteins PIN3, ABCB19, and AUX1, were not observed in any endomembrane compartments. As all of these proteins are trafficked and recycled, and the scd1-1 mutation most strongly affects PIN1 and PIN2 and dependent physiological processes, there appears to be selectivity of SCD1 targets toward PIN2 and PIN1, which is consistent with other studies that reveal distinct regulatory and trafficking machinery between auxin transporters , 1.5% sucrose (w/v), and 0.05% MES (w/v); pH adjusted to 6.0 with 1M KOH. The seeds were placed in the dark at 4°C for 48 h before being moved to the permissive (18°C) or the restrictive (25°C) temperatures. The seedlings were grown vertically under continuous light at an intensity of 100 μmol m–2s–1 for 5 or 7 days at the specified temperature.Seeds were sterilized  for 2 min, followed by a 95% ethanol rinse. The culture medium was 0.8% (w/v) agar , 1X MS nutrients , vitamins  and scd1-1 seedlings were assayed at 10 days for both 18°C and 25°C to more closely match the length of scd1-1 and Col(g). An agar droplet (1.25% agar w/v) containing 1 μM tritiated IAA was placed at the root shoot junction and incubated at 18 or 25°C for 10 min. The seedlings were transferred to a new agar plate and exposed to a “chase” droplet of unlabeled 1 μM IAA. After 50 min, 2 mm sections were excised beginning at the base of the droplet toward the root tip. Three to six segments were recovered from all plants, and each segment was counted separately, and IAA levels quantitated using a Beckman LS6500 liquid scintillation counter. The average and SE of 19–25 seedlings is reported in all transport assays.Pulse-chase rootward  IAA transport assays were performed using a previously published method  in Col(g3H-IAA) to root tips  . Dropletscd1-1 seedlings grown at 18°C were placed in diffuse light at 20–22 μmol m–2 s–1 and re-oriented 90°. Images were captured for low temporal resolution analysis at time 0 and at 2 h intervals until 8 h after re-orientation, with a final image 24 h after re-orientation. Images were captured using an Epson Perfection 3200 Photo Perfection flatbed scanner  with 16-bit grayscale and 800 dpi resolution. Images were exported to ImageJ to measure degree of curvature.Five-day old Col(g) and The gravitropic root response was also observed using high temporal resolution imaging using an AVT Marlin camera . Root curvature after gravity stimulation was measured by a MATLAB based custom image analysis software  and is tscd1-1 seedlings crossed with the proDR5-GFP construct were imaged after roots were reoriented 90° relative to the gravity vector. Seedlings were placed on agar plates on the 5th day after growth at 18 or 25°C. After 1 h, seedlings were placed in diffuse light at 20–22 μmol m–2s–1 and reoriented 90°. Images were captured at time 0 and at 2 h intervals until 8 h after re-orientation. Seedlings were stained with propidium iodide (PI) to visualize root structure and mounted in dH20 on slides. The root tips were visualized with the 40x objective of a Zeiss 710 LSCM using the argon laser at an excitation wavelength of 488nm and a pinhole of 1.0 AU. GFP emission was collected between 492 and 547 nm, while PI was collected at 567–735. All images were collected at identical gain and laser power and exported to Adobe Photoshop for analysis. Representative images of more than 5 roots at each time point and from each genotype were shown.Col(g) and scd1-1 seedlings containing PIN2-GFP, PIN1-GFP, PIN3-GFP, ABCB19-GFP, and AUX1-YFP (protein fusions driven by endogenous promoters) were grown for 7 days at a constant 18 or 25°C. Seedlings were mounted in dH2O and maintained at the indicated temperatures using a Julabo F25 temperature stage . Images were captured using a 63x water objective with or without a 1.5x optical zoom on a Zeiss 710 LSCM, using the argon laser for excitation at 488 nm with a pinhole of 1.0AU. PIN2-GFP, PIN1-GFP, PIN3-GFP, and MDR1-GFP were imaged with an emission spectrum of 500–525 nm, while AUX1-YFP was imaged with an emission spectrum of 519–621 nm.Col(g) and scd1-1 were grown at 18°C for 7 days. Seedlings were transferred to microscope slides and then placed on the temperature-controlled stage at 25°C. Samples were imaged as described above at various times after transfer. For each reporter construct more than 5 roots were analyzed at each treatment, with representative images shown.For temperature shift experiments, wild-type and 2O and imaged at various times after staining. The GFP and FM4-64 signals were visualized using a multi-track setting, as described above. No FM4-64 fluorescence was detected in the GFP channel. The FM4-64 signal was imaged using 80% laser power, a pinhole of 1.0 AU, 510 gain, and an emission spectrum of 632–759 nm. No GFP signal was detected in the FM4-64 channel. Individual images for each fluorophore and an overlay are shown with magenta for FM4-64, green for PIN2-GFP, and white for any pixels with both FM4-64 and PIN2. The red signal in these images was converted to magenta in Photoshop, changing the hue saturation. To better identify spatial colocalization independent of intensity, a thresholding function was applied to individual channels in Photoshop or in the Zeiss Zen colocalization module. We then prepared a thresholded overlay in which pixels are indicated as magenta for FM4-64, green for PIN2-GFP, and white for any pixels with both FM4-64 and GFP. To examine the change in colocalization of PIN2-GFP and FM4-64 over time, the number of PIN2-GFP, FM4-64, or both, signals in each cell with a PIN2-GFP labeled endomembrane compartment was quantified, and the percentage was determined for 740 cells from 20 roots. In the temperature shift experiments, scd1-1 seedlings grown at 18°C for 7 days were transferred to 5 mM FM4-64 for 5 min, rinsed briefly, and then imaged at intervals of 2 min on a temperature-controlled stage at 25°C.Seedlings grown at 25°C were incubated in 5 μM FM4-64 to visualize endosomes  for eithscd1-1 seedlings containing PIN2-GFP were crossed with fluorescently labeled endomembrane marker lines. These constructs and their localization patterns are summarized in Col(g) and Lines containing both PIN2-GFP and other RFP reporters were examined in dual channel mode as described above. PIN2-GFP SYP61-CFP transgenics were imaged using spectral imaging and linear unmixing at an excitation wavelength of 458 nm and a 464–648 nm filter.All datasets generated for this study are included in the article/CG and JI designed and completed the experiments and wrote the manuscript. TF, CM, DL, and KM performed the experiments and edited the manuscript. GM designed the experiments, wrote, and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nivolumab showed improvement in overall survival (OS) in ATTRACTION-2, the first phase 3 study in patients with gastric/gastroesophageal junction (G/GEJ) cancer treated with ≥ 2 chemotherapy regimens. The 2-year follow-up results of ATTRACTION-2 are presented herein.ATTRACTION-2 was a randomized, double-blind, placebo-controlled, phase 3 trial . The median (min–max) follow-up period was 27.3 (24.1–36.3) months. The primary endpoint was OS. A subanalysis of OS was performed based on best overall response and tumor-programmed death ligand-1 (PD-L1) expression status.P < 0.0001). A higher OS rate was observed in the nivolumab vs placebo group at 1 (27.3% vs 11.6%) and 2 years (10.6% vs 3.2%). The OS benefit was observed regardless of tumor PD-L1 expression. Among patients with a complete or partial response (CR or PR) in the nivolumab group, the median OS (95% CI) was 26.6 (21.65—not applicable) months; the OS rates at 1 and 2 years were 87.1% and 61.3%, respectively. No new safety signals were identified.Overall, 493 of 601 screened patients were randomized (2:1) to receive nivolumab (330) or placebo (163). OS  was significantly longer in the nivolumab group (5.26 [4.60–6.37] vs 4.14 [3.42–4.86] months in placebo group) at the 2-year follow-up  contains supplementary material, which is available to authorized users. Over 1,000,000 new cases of gastric/gastroesophageal junction (G/GEJ) cancer were reported in 2018 , disease control rate , and BOR . Tumor responses were assessed with computed tomography (CT) or magnetic resonance imaging (MRI) after each treatment cycle for first ten cycles and after every two treatment cycles thereafter until discontinuation of study treatment or the initiation of the poststudy treatment. Tumors were assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines version 1.1 .Adverse events (AEs) were evaluated using the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0  during tTumor tissue collection was not mandatory, and exploratory analysis of PD-L1 expression (PD-L1 positivity: 1% or more of tumor cells) was performed by a central laboratory using immunohistochemistry  on the available tumor samples. Exploratory subanalysis of OS was performed in patients with CR + PR, SD, and PD.Additionally, an exploratory landmark analysis was performed in patients who had SD at the first 6-week assessment. Patients with SD at the first 6-week assessment were categorized into the following three groups based on the tumor growth rate at 6 weeks: group 1 (− 30% < and ≤  − 5%), group 2 (− 5% < and <  + 5%), and group 3 (+ 5% ≤ and <  + 20%); OS curves were generated from 6 weeks onwards. The tumor growth rate was calculated as a change in tumor volume from baseline in the 6-week period. Since no definitive cutoff value was specified for this categorization, this landmark analysis was exploratory.Sample size estimation has been described previously . OS and Overall, 601 patients were screened, of whom 493  were randomized in ATTRACTION-2. The safety assessment population comprised 491 patients , and the response assessment population comprised 399 patients with measurable lesions . Further details of patient disposition have been reported previously  vs 109 [67.7%]), worsening of clinical symptoms judged as PD (59 [17.9%] vs 39 [24.2%]), onset of grade ≥ 2 interstitial lung disease (5 [1.5%] vs 0 [0%]), physician discretion (13 [3.9%] vs 3 [1.9%]), treatment withheld longer than 6 weeks due to AEs (7 [2.1%] vs 1 [0.6%]), and other reasons (27 [8.2%] vs 19 [11.8%]).Following study treatment discontinuation, 53.6% (177/330) and 47.2% (77/163) of patients in the nivolumab and placebo groups, respectively, received subsequent anticancer treatment . Among all patients, 109 (33%) patients in the nivolumab group and 37 (23%) patients in the placebo group continuously received the study treatment after being judged as having PD as per RECIST version 1.1. In total, six (1.8%) patients in the nivolumab group and two (1.2%) patients in the placebo group received immune checkpoint inhibitors as subsequent therapy.P < 0.0001; Fig. The median OS (95% CI) in the nivolumab vs placebo group was 5.26 (4.60–6.37) vs 4.14 (3.42–4.86) months at the 2-year follow-up. The OS rate was longer in the nivolumab group than in the placebo group throughout the study period [P < 0.0001; Fig. The median PFS (95% CI) in the nivolumab group compared with the placebo group was 1.61 1.54–2.30) vs 1.45 (1.45–1.54) months at the 2-year follow-up. The PFS rate was higher in the nivolumab group than in the placebo group after approximately 2 months of treatment initiation throughout the study period [.30 vs 1.The ORR was greater in the nivolumab group than in the placebo group, with a CR or PR observed in 32 patients  compared with no patients, respectively, at the 2-year follow-up . Of note, no CRs and 30 PRs had been observed at the initial follow-up  [Among patients with a CR or PR in the nivolumab group, the median (95% CI) OS was 26.6 (21.65—not applicable) months; the OS rates at 1 and 2 years were 87.1% and 61.3%, respectively. No patient in the placebo group had a CR or PR  OS in patients with PD-L1-positive tumors was 5.22 (2.79–9.36) months in the nivolumab group and 3.83 (0.79–4.96) months in the placebo group . In patients with PD-L1-negative tumors, median (95% CI) OS was 6.05 (4.83–8.61) months in the nivolumab group and 4.19 (3.02–6.93) months in the placebo group . The OS benefit was observed regardless of PD-L1 expression status as reported previously [Among patients in whom response could be evaluated, the exploratory landmark analysis showed that in patients with SD at the first 6-week assessment, difference in the median OS between the nivolumab and placebo groups was 8.81, 3.55, and 3.15 months in the tumor growth rate group 1 (− 30% < and ≤  − 5%), group 2 (− 5% < and <  + 5%), and group 3 (+ 5% ≤ and <  + 20%), respectively. The OS rate and curves in the nivolumab group were slightly better than those in the placebo group across the three tumor growth rate groups .Safety analyses were performed in 330 patients in the nivolumab group and 161 patients in the placebo group who received one or more doses of nivolumab. All-cause AEs of any grade were reported in 301 91.2%) of 330 patients in the nivolumab group and 135 (83.9%) of 161 patients in the placebo group. TRAEs of any grade were reported in 142 (43.0%) patients in the nivolumab group and 43 (26.7%) patients in the placebo group, including 39 (11.8%) and seven (4.3%) patients with grade 3–4 TRAEs, respectively. Serious TRAEs were reported in 38 (11.5%) of 330 patients in the nivolumab group and eight (5.0%) of 161 patients in the placebo group. Most patients experienced onset of TRAEs of special interest within 3 months of starting nivolumab: skin, gastrointestinal, hepatic, and endocrine TRAEs were most commonly experienced at 3 months and tended to abate over time. The incidence rates of TRAEs of special interest were comparable at 6 months, 1 year, and 2 years. No major late-onset TRAEs were observed  on nivolumab had SD. Patients with SD at 6 weeks had a range of tumor growth rate from − 30 to + 20%, meaning either a slight decrease or a slight increase. We performed an exploratory analysis that assessed OS among SD patients by subgrouping them based on the tumor growth rate at the first assessment  to examine whether or not the continued use of nivolumab could provide clinical benefit to SD patients even after a slight increase in tumor size. When categorized by three tumor growth rate groups  in these patients with SD at 6 weeks, the OS rate/curves in the nivolumab group were higher than those in the placebo group across the three tumor growth rate groups. Comparing OS in the subset showing SD at 6 weeks might provide further insights into the efficacy of nivolumab. Patients with SD in the placebo group had more indolent and slow-growing tumors compared with patients with PD, but some of the patients with SD in the nivolumab group might have had aggressive tumors whose growth could be inhibited by nivolumab. Furthermore, in the phase III trial of nivolumab in non-small cell lung cancer, discontinuation of nivolumab after disease control for 1 year resulted in poor prognosis compared with its continuation . It is sAll five of the patients with PD at initial response assessment who survived longer than 2 years received post-progression therapy, and three of them continued nivolumab beyond PD. Only one patient showed tumor shrinkage with nivolumab beyond PD. While this study allowed continuation of nivolumab beyond PD conditionally, the clinical significance of this treatment is not clear., the results of OS by BOR should be interpreted with caution because clinical significance of continuing nivolumab should be confirmed in a randomized trial. Furthermore, other prognostic factors, including natural tumor growth kinetics , may have influenced the outcome in patients with SD.OverallThe efficacy of nivolumab was similar to and sustained from the 1-year follow-up as demonstrated by continued clinically meaningful improvements in OS and PFS at the 2-year follow-up compared with placebo. The long-term survival benefit of nivolumab was most evident in patients with CR or PR than in those with SD or PD. Even among patients with SD categorized by tumor growth rate, nivolumab offered a longer median OS than placebo, suggesting that nivolumab can be continued even after a small increase in tumor size within SD. However, these observations will need to be validated in future studies by evaluating the use of nivolumab in beyond-PD cases. The safety profile was similar to that at the 1-year follow-up, and no major late-onset TRAEs of special interest were observed; however, continual monitoring of AEs was necessary.Supplementary file1 (DOCX 1539 kb)Below is the link to the electronic supplementary material.


HUVEC cells pre-treated with TSG for 24 h were exposed to H2O2 treatment. TSG significantly decreased H2O2-induced cellular senescence, as indicated by reduced senescence-associated β-galactosidase  positive staining, the proportion of cells in the G1 phase, cell apoptosis, p21, and plasminogen activator inhibitor-1 (PAI-1) expression. Moreover, TSG promoted Sirtuin 1 (SIRT1) expression. When SIRT1 was inhibited by EX527 or SIRT1 siRNA, the effect of TSG is diminished according to the increased proportion of cells in the G1 phase, cell apoptosis, p21, and PAI-1 expression. Overall, our study established TSG as an anti-senescence compound that exerts its protective action by regulating SIRT1 expression.This study aimed to explore the effects of 2,3,5,4’-tetrahydroxy-stilbene-2-O-β-d-glucoside (TSG) on the senescence of human umbilical vein cells (HUVEC) induced by hydrogen peroxide (H Wang etThis study aimed to demonstrate whether TSG can relieve the HUVEC senescence and investigate how SIRT1 functions in the process of HUVEC senescence when TSG is present.The HUVEC were purchased from the Institute of Biology, Chinese Academy of Sciences. The 2,3,5,4’-Tetrahydroxystilbene-2-O-β-D-Glucoside was purchased from Sigma company, American (lot: M48818078). And TSG was dissolved in sterile water.2. HUVECs were pretreated with various concentrations of TSG (20 and 40 μg/ml) 24 h. Then HUVECs were treated with H2O2 (0.2 mM) 2 h and cultured in normal medium for 24 h later. As for EX527, the specific inhibitor of SIRT1, were added 40 μM for 24 h before pretreating with TSG.HUVEC were cultured in RMPI 1640 medium  containing 10% heat-inactivated fetal bovine serum  and 1% penicillin-streptomycin at 37°C in 5% COThe MTS assay was used to assess cell viability. Before each experiment, HUVEC  were seeded in 96-well microtiter plates and treated. Subsequently, 20 μl MTS solution was added to each well, and the plates were incubated for 2 h at 37°C. The absorbance was measured at 490 nm and used to calculate the relative ratio of cell viability.Senescence was assessed by β-galactosidase staining. Cells were washed in PBS and then fixed with fixation fluid for 15 min at room temperature. After washing three times, cells were incubated at 37°C overnight with dyeing liquor. At the end of the incubation, five random fields were counted per dish to assess the percentage of senescence-associated beta-galactosidase  positive cells.The cell cycle was detected by flow cytometry-based on propidium iodide (PI) staining. Cells were collected and fixed overnight with 70% alcohol at 4°C. The cells were centrifuged to remove the alcohol and washed twice with cold PBS. Next, the PI stain was added, and the cells were protected from light for 30 min at 4°C. Cell cycles were analyzed, and the percentage of cells in the G1 phase was recorded.Cell apoptosis analysis was performed using the Annexin V-FITC Apoptosis Detection Kit (Beyotime). Briefly, after experimental treatments, the cells were washed twice with cold PBS and resuspended in 100 μl binding buffer, followed by incubation with 5 μl Annexin V-FITC and 10 μl PI at room temperature for 15 min. A total of 10,000 cells were collected and analyzed by flow cytometry  equipped with a Cell Quest software (BD Biosciences).HUVEC were fixed for 30 min at room temperature in 4% paraformaldehyde. Next, HUVEC were treated with PBS containing 0.5% Triton X-100 for 10 min and were then blocked with 5% BSA for 1 h. Cells were subsequently incubated with SIRT1 antibody  overnight at 4°C and then incubated with anti-rabbit secondary antibody at room temperature for 2 h. Chromosomes were stained with DAPI for 5 min. The HUVEC were mounted on glass slides and examined with a laser scanning confocal microscope (Zeiss LSM 880).Briefly, proteins were extracted with radio immunoprecipitation assay (RIPA) solution, and the concentration was measured with a bicinchoninic acid (BCA) kit . Protein samples were separated by SDS-PAGE using a 10% polyacrylamide gel. Then, membranes were exposed to anti-SIRT1 , anti-p21 , anti-PAI-1 , and anti-GAPDH  overnight at 4°C. The membranes were washed  in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) and then incubated with the corresponding secondary antibody.®Prime Ex Taq TM II (Tli RNaseH plus)  at 95°C for 2 min, and then 56°C for 30 s, 72°C for 30 s for 40 cycles. The primer sequences used in this study were as follows: SIRT1 F: ACTTCAGGTCAAGGGATG, and R: CACTGCACAGGCACATAC.Total RNA was extracted with TRIzol reagent. The complementary DNA was synthesized using Prime Script TM RT Master Mix  for reverse transcription PCR with conditions of 37°C for 15 min, 85°C for 5 s, and storage at 4°C. The Real-time fluorescence quantitative PCR was performed in duplicate using SYBR5 cells/plate and cultured for 24 h. Then, the cells were transfected with SIRT1 siRNA (30 pmol) 12 h using Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s protocols.Cells in the exponential phase of growth were plated in six-well plates at 2 × 10post hoc test was performed. The results were considered to be statistically significant when p <0.05.All experiments were performed independently in triplicate. The results are presented as mean ± SD calculated by GraphPad Prism. For multiple comparisons, one-way ANOVA followed by Tukey 2O2-treated HUVEC  significantly increased SIRT1 mRNA and protein expression. In order to more directly observe the effect of TSG on the expression of SIRT1 in HUVEC, immunofluorescence experiments were performed showing that TSG can significantly increase the fluorescence intensity associated with SIRT1 in senescent HUVEC, suggesting that TSG may block H2O2-induced HUVEC senescence via SIRT1.SIRT1 was usually associated with longevity. So, we speculated that it may become a target of anti-senescence drugs. As shown in 2O2-induced HUVEC senescence by promoting the expression of SIRT1, we used EX527, a specific inhibitor of SIRT1. EX527 did not induce significant cytotoxicity in normal HUVEC  proteins increased, and TSG had a little protective effect on HUVEC senescence. The expression level of PAI-1 and p21 proteins in SIRT1 siRNA group were lower than those in H2O2 group, which was consistented with the results in the literature ; and the National Natural Science Foundation of China (No.81630105).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The pristine material contained a composite layered structure composed of Ni-free and Ni-doped Li2MnO3 and LiMO2  nanoscale domains, and Li ions were sequentially and inhomogeneously extracted from the composite structure. Delithiation from the LiMO2 domain was observed in the potential slope region associated with the Ni2+/Ni4+ redox couple. Li ions were then extracted from the Li2MnO3 domain during the potential plateau and remained mostly in the Ni-doped Li2MnO3 domain at 4.8 V. In addition, structural transformation into a spinel-like phase was partly observed, which is associated with oxygen loss and cation migration within the Li2MnO3 domain. During Li intercalation, cation remigration and mixing resulted in a domainless layered structure with a chemical composition similar to that of LiNi0.25Mn0.75O2. After the structural activation, the Li ions were reversibly extracted from the newly formed domainless structure.Lithium- and manganese-rich layered oxides (LMRs) are promising positive electrode materials for next-generation rechargeable lithium-ion batteries. Herein, the structural evolution of Li Rechargeable lithium ion batteries (LIBs) have been widely used as a power source for portable devices and currently the global market for electric vehicles (EVs) demands higher power, higher energy density, longer life, and lower cost batteries. A significant amount of effort has been dedicated to the development of improved battery materials, with recent studies investigating lithium- and manganese-rich layered oxides (LMRs), which are preferred candidate materials for the next-generation LIB positive electrodes due to their high reversible capacities of ≥200 mA h g–1 at 2.0–4.8 V10. In contrast, several drawbacks have been reported including capacity fading and voltage decay during long-term charge–discharge cycles14. The structural factors resulting in these problems must be solved to develop stable and high power batteries.The world is moving towards electrification as CO×)/3(1–2MxMnx)/3 in single-phase notation or as xLi2MnO3·(1–x)LiMO2 in composite notation. Based on single-phase notation, the crystal structure can be indexed with a space group of C2/m, where the structure is homogeneous and Li ions occupy the Li layer and part of the transition-metal (TM) layer with intralayer ordering between the Li and TM ions. In contrast, the composite notation can be expressed as a mixture of C2/m and R–3 m structures, where Li2MnO3  and LiMO2 exhibit common d spacing. These two phases are dispersed as nanoscale domains over the entire structure15. Unfortunately, X-ray diffraction (XRD) studies provided no clear indication of which structural model appropriately represents the real structure. Many atomic column observations using advanced scanning transmission electron microscopy (STEM) have provided evidence of two structural domains in the composite structure19, while other reports showed a homogeneous atomic column, suggesting a single-phase structure22. Solid-state nuclear magnetic resonance (NMR) spectroscopy is sensitive to cation substitution in the first and second cation coordination shells, allowing the local structure to be examined at a length scale of <5 Å in diameter. Grey et al. reported the nonrandom cation distribution around Li ions in Li[Li×)/3(1–2MxMnx)/3(2–]O2 via 6Li magic-angle spinning (MAS) NMR analyses, implying a composite nature instead of homogeneous solid solution25.LMRs exhibit a layered rock-salt structure commonly represented as Li O2  compared to S = 3/2 for Mn4+, yielding weaker hyperfine interactions, the 7Li signals in the Ni-doped Li2MnO3 (Li2[Ni1/6Mn5/6]O3-like) domain appeared at lower frequencies (553 and 1331 ppm) compared to those in the Ni-free domain (749 and 1514 ppm)64. The 791 ppm component was tentatively assigned to the 7Li signal from the Ni-containing LiMO2 domain due to the lack of the corresponding 7Li signal from the TM layer. Some assignments differed from those in a recent study39, where the peaks at 576 and 946 ppm (corresponding to the peaks at 553 and 791 ppm observed herein) were assigned oppositely. The remaining 352 ppm component was not observed in previous studies64, and was tentatively ascribed to Li ions in the Li layer of the Li2[Ni2/6Mn4/6]O3-like domain. This contribution is negligible and is merged with the discussion of the 553 ppm peak below. Finally, the sharp signal at 0 ppm arises from impurity phases such as Li2CO3 and organic lithium salts.Figure 1 T Fig. . The 7Lints Fig. . There a O2 Fig. . The pea2MO3 and LiMO2-like structure were alternately stacked over several nm . Therefore, the bulk chemical composition of the pristine material was re-expressed as Li[Li0.17Ni0.21Mn0.59]O2  including the Ni and Mn valence states estimated from previous XAS results 49. The composition and ratio of the Ni-free and Ni-doped Li2MnO3 (Li1.33Mn0.67O2) and LiMO2 domains were estimated as 0.28Li1.37Mn0.63Oδ)2(– (similar to Li1.33Mn0.67O2), 0.28Li1.26Ni0.11Mn0.57O2, and 0.43LiNi0.41Mn0.57O2, considering the experimental 7Li signal ratio LiTM/LiLi of each domain , and cation vacancies as well as the assumption that the Ni ions are tetravalent in the Ni-doped Li2MnO3-like domain with Ni:Mn = 1:5 . The estimated phase ratio is comparable to the composite notation of Li1.2Ni0.2Mn0.6O2, 0.6Li1.33Mn0.67O2·0.4LiNi0.5Mn0.5O2. It should be emphasized that the Ni-doped Li2MnO3-like domain, Li1.26Ni0.11Mn0.57O2, is a dominant component in the material, acting as a boundary buffer phase between the Li2MnO3 and LiMO2 domains39. The composition and ratio of these domains differed from those estimated in ref. 39 with a similar bulk composition, Li1.2Ni0.18Mn0.61Mg0.01O2, mostly due to different peak assignments. It should be noted that the composite structure of differently composed domains in Li1.2Ni0.2Mn0.6O2 may introduce chemical inhomogeneity within a nanoscale particle. The inhomogeneous Ni distribution relative to Mn and O is highlighted in the energy-dispersive X-ray spectrometry (EDS) mapping images of a single particle , was used instead of the ICP-AES-based composition, Li1.25Ni0.20Mn0.55Oδ2–, or the ideal composition, Li1.2Ni0.2Mn0.6O2, for consistency. Total 7Li signals decreased in intensity during charging. It should be noted that the Li contents at 50 and 100 mA h g–1 were significantly underestimated compared to those calculated from the delivered current densities and similar behaviors were reported for LiCoO2 and LiNi0.5Mn1.5O466. This abrupt intensity reduction during the early stages of delithiation arises from the localized nature of the electrons on paramagnetic ions and nearby Li+ ions, causing rapid relaxation of all nearby 7Li nuclear spins before signal acquisition. In contrast, the Li content in the sample at 4.8 V was slightly overestimated, indicating that the delivered charging capacity includes some contribution from side reactions such as electrolyte decomposition at high potentials. During discharging, the Li contents increased almost linearly. Figure 0.17Ni0.21Mn0.59]O2. The LiTM/LiLi ratio remained almost constant during charging, but decreased gradually during discharging , Li extraction is electronically compensated by the valence increase of Ni in the LiMO2 domain. It should be noted that some 7Li signals were lost in the Ni-free and Ni-doped Li2MnO3 domains over the potential slope region -STEM images67. The structure of Li1.2Ni0.2Mn0.6O2 after the initial cycle changed to a domainless single phase LiMO2, although some Li2MnO3 domain remained, indicating that irreversible structural changes occur during the initial charge–discharge process.The Li content in each domain during the 1ove Fig. . Therefoon Figs.  and S7. ppm Fig. , #5, sug8 V Fig. , delithi7Li signal at 1514 ppm is relatively sharp and well separated from other components, allowing the peak position changes to be confirmed with high confidence  to Ni4+ (S = 0), reducing the Fermi contact interaction and shifting the peak position to lower frequencies. In contrast, no peak shift was observed over the potential plateau region, indicating that the TM ions were not involved in charge compensation. During the discharging process, the peak position returns, to some extent, to higher frequencies due to TM ion reduction. These changes are qualitatively consistent with that of the a lattice parameter.The nce Fig. . Over th2MO3 and LiMO2 domains to a single-phase (domainless) structure. This is associated with cation mixing  during the formation of a spinel-like structure at a high potential. Upon back-transformation to a layered structure during discharging, the TM ions move back to positions different from their original positions in the TM layer, and the TM ions are uniformly dispersed, resulting in a domainless structure.Therefore, it can be concluded that the irreversible potential plateau in the initial charge–discharge cycle corresponds to a structural change  from a composite structure of Li1.2Ni0.2Mn0.6O2 after the initial charge–discharge cycle will be briefly discussed. The charge–discharge profiles for the 2nd and 20th cycles are shown in Fig. th cycle  are quite similar to those of the 1st cycle (#5 and 7). Even at the 20th charge–discharge cycle, the 001 diffraction peak of the charged sample was split  of layered structure phase and the increase in domain size  of spinel phase. In addition, a small shoulder at the lower 2θ side of the 001 diffraction peak in the 20th discharged sample may be a remnant peak of the layered structure observed in the charged state. These results indicate that the structural changes are reversible after structural activation during the initial cycle. This structural stability is significantly different from that of Li2MnO3, which changes from a layered structure to a highly disordered spinel-like structure in 20 cycles56. It should be noted that the spinel-like phase was observed at 4.8 V in the 20th cycle, although the Li2MnO3 domain completely disappeared, as discussed below. Therefore, this spinel-like structure results from the domainless layered structure at the high voltages68. Figure st, 2nd, and 20th cycles. The domain structure in the pristine material changes to a domainless structure after the 1st discharge and both the a- and c-axes were correspondingly expanded. The lattice parameters further increased for the 2nd and 20th discharged materials. The extent of TM ion migration (to the Li layer) and oxygen site occupancy remained nearly constant after the 1st cycle 43, while these soft XAS spectra include some contributions from the active material surface degradation. The present results suggest that Ni ions become redox inactive at higher voltages (> ~3.2 V for Ni2+/Ni4+ redox couple), and Mn ions become redox active at lower voltages (<~3.2 V for Mn3+/Mn4+ redox couple) during repeated cycles. This is involved in voltage fading behavior shown in Fig. Figure ly Table . The avely Table , which i7Li pj-MATPASS spectra of the 1st, 2nd, and 20th charged and discharged samples, where comparison of the charged  and discharged samples  shows no significant differences in the spectra, except for consumption of the residual signals of the Li2MnO3 component at the 20th cycle. Therefore, the Li ions were reversibly extracted and inserted into the domainless layered structure after the initial cycle. Broad signals centered at 585 and 630 ppm were observed in the 20th charged and discharged materials, respectively. The higher frequency isotropic shift corresponds to the lower average oxidation state of TM ions, with higher spin numbers of S = 2 and 1  compared to S = 3/2 and 0 . The Li contents estimated from the signal intensities of the 20th charged and discharged samples were slightly overestimated relative to that calculated from the charging and discharging capacities , although the Li content calculated from the capacity at 4.8 V should be empty. These results indicate that the long-cycled charge–discharge capacity includes significant contributions from accumulative side reactions and that NMR spectroscopy is a reliable tool for directly measuring the Li content in active materials.Figure ies Fig. . Li ions1.2Ni0.2Mn0.6O2 was reexamined during charge–discharge cycles. It was confirmed that the pristine material contains a composite layered structure composed of Li2MO3 and LiMO2 nanoscale domains using electron microscopy (HAADF-STEM). Solid-state NMR further provided a quantitative information about the composite structure , which consists of pure Li2MnO3, Li2[Ni1/6Mn5/6]O3, and LiO2 nanoscale domains. We found that Li ions are sequentially and inhomogeneously extracted from the composite structure. Li ions were deintercalated from the LiO2 domain in the potential slope region during initial charging, where the Ni ions were redox active for charge compensation. Subsequently, Li ions were preferably extracted from both the Li and TM layers in the Li2MnO3 domain in the potential plateau region at a high voltage, where the charge was compensated by oxygen anions by formation of ligand holes, peroxide-like moieties, or O2 release. Li ions mostly remained in the Li layer of the Li2[Ni1/6Mn5/6]O3 domain at 4.8 V. The XRD profiles indicated a reversible phase separation into layered rock-salt and spinel-like nanodomain structures at 4.8 V, where the latter phase is associated with TM ion migration from the TM layer into the vacant Li layer in the Li2MnO3 domain. During Li intercalation into the structure, cation remigration and mixing formed a domainless layered structure, and the active material became a single Li[Ni0.25Mn~0.75]O2 phase with a small content of remnant Li2MnO3 domain. After the 1st charge–discharge cycle, when irreversible structural activation occurred, the bulk structure showed reversible Li extraction accompanied by reversible lattice changes and phase separation into layered and spinel-like phases at high potentials. These results provide a new insight into the delithiation/lithiation behavior of nanoscale composite-structured LMRs, which further serve as understanding of the capacity and voltage fading mechanism relating to structural degradation of these materials.Based on the composite structure concept of LMRs, the delithiation behavior of Li1.2Ni0.2Mn0.6O2 was synthesized via solid-state reaction at 900 °C for 12 h with the starting materials, LiOH · H2O, Ni2CO3  and MnCO3 43. From scanning electron microscopy (SEM) observations, the average particle size was estimated to be ~5 μm, composed of an aggregate of small crystallites (~200 nm in diameter). The chemical composition of the obtained material was determined to be Li1.25Ni0.20Mn0.55Oδ2– (δ ≈ 0.07) via inductively coupled plasma-atomic emission spectrometry  and iodometric titration measurement that estimates the average oxidation state of the transition metals.Li56 The electrode was pressed to a thickness of 30–35 μm and foils of metallic lithium  were used as counter and reference electrodes. These components were assembled with the Celgard 2500 separator and soaked in an electrolyte solution, and sealed in an aluminum-coated laminate-type cell in an Ar-filled glove box. The electrolyte solution used herein was 1 M LiPF6 solution dissolved in anhydrous ethylene carbonate (EC) and ethylmethyl carbonate (EMC) at a volumetric ratio of 3:7 .A positive electrode was prepared using a mixture of the active material, acetylene black (Denki Kagaku Kogyo), and polyvinylidene difluoride  at a weight ratio of 80:10:10 spread onto an aluminum foil with 1-methyl-2-pyrrolidone (NMP) and dried at 80 °C under vacuum overnight.+ at a current rate of 20.5 mA g-1 56. The cells were carefully disassembled at the desired charge/discharge states in the glove box and rinsed with dimethyl carbonate (DMC) to remove any electrolyte solution residue.The electrochemical measurements were performed at 50 °C on an automatic cycling and data recording system . The cells were galvanostatically cycled between 2.0 and 4.8 V vs. Li/Liϕ borosilicate glass capillaries in the 2θ range of 4.0–52.7° with Δθ of 0.0025° at the BL28XU beamline in SPring-8 . An incident radiation of λ = 0.4997 Å was used, which was calibrated with the lattice parameters of CeO2. The Rietveld refinements of the crystal structure parameters were performed using Jana200669.Synchrotron-radiation XRD (SR-XRD) measurements were performed using powdered samples sealed in 0.5 mmII,III-edge, and O K-edge spectra were acquired in partial fluorescence yield (PFY) mode. Mn LII,III-edge spectra were also acquired in inverse partial fluorescence yield (IPFY) mode to account for self-absorption effects70. The PFY/IPFY spectra are (relatively) bulk-sensitive with a probing depth of up to ~200 nm71.Soft XAS measurements were performed at beamline BL-11 in the SR Center, Ritsumeikan University . The samples were placed into a sample holder in the glove box and transferred using a transfer vessel to the high vacuum sample chamber without exposure to the air. The Ni, Mn L7Li MAS NMR spectra were acquired using an ECA-600 spectrometer (JEOL RESONANCE Inc.) at a magnetic field of 14.1 T with a wide-bore T3 MAS probe (Agilent Technologies). The powder samples were packed into 1.6 mmϕ MAS ZrO2 rotors with airtight caps in a glove box and spun at a spinning rate of 30 kHz during the experiments. The practical temperatures of the spinning samples at 30 kHz were estimated to be ~60 °C due to frictional heating. To obtain high resolution spectra, the pj-MATPASS technique40 was used with a π/2 pulse width of 1.1 μs and a relaxation delay of 0.1 s. All spectra were referenced to a 1 M LiCl solution at 0.0 ppm. The signal intensities were normalized by the sample weight in the rotors.Supplementary information.


Histone methylation is a context-dependent modification that regulates gene expression, and the trimethylation of histone H3 lysine 27 (H3K27me3) usually induces gene silencing. Overcoming colorectal cancer (CRC) chemoresistance is currently a huge challenge, but the relationship between H3K27me3 modification and chemoresistance remains largely unclear. Here, we found that H3K27me3 levels positively correlated with the metastasis-free survival of CRC patients and a low H3K27me3 level predicted a poor outcome upon chemotherapeutic drug treatment. Oxaliplatin stimulation significantly induced the expression of H3K27 lysine demethylase 6A/6B (KDM6A/6B), thus decreasing the level of H3K27me3 in CRC cells. Elevation of H3K27me3 level through KDM6A/6B depletion or GSK-J4 (a KDM6A/6B inhibitor) treatment significantly enhanced oxaliplatin-induced apoptosis. Conversely, when inhibiting the expression of H3K27me3 by EPZ-6438, an inhibitor of the histone methyltransferase EZH2, the proportion of apoptotic cells remarkably decreased. In addition, the combination of GSK-J4 and oxaliplatin significantly inhibited tumor growth in an oxaliplatin-resistant patient-derived xenograft model. Importantly, we revealed that oxaliplatin treatment dramatically induced NOTCH2 expression, which was caused by downregulation of H3K27me3 level on the NOTCH2 transcription initiation site. Thus, the activated NOTCH signaling promoted the expression of stemness-related genes, which resulted in oxaliplatin resistance. Furthermore, oxaliplatin-induced NOTCH signaling could be interrupted by GSK-J4 treatment. Collectively, our findings suggest that elevating H3K27me3 level can improve drug sensitivity in CRC patients. Colorectal cancer (CRC) ranks third in morbidity and fourth in mortality of all human cancers worldwide . The incHypothetical drug resistance mechanisms include inefficient cellular drug accumulation , increasHistone methylation modification is an important process involved in the regulation of gene expression, and this modification is controlled by histone methyltransferases and demethylases. In general, histone H3 lysine 27 trimethylation (H3K27me3) leads to gene repression, whereas histone H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 36 methylation (H3K36me2/me3) promote gene transcription . It has in vivo and in vitro. Additionally, the effect of H3K27me3 modification on chemoresistance is achieved by targeting NOTCH2 expression, thus modulating the expression of stemness-related genes in CRC cells.Here, we demonstrated that low H3K27me3 levels could result in CRC resistance to chemotherapeutics and predict poorer prognoses in patients with CRC. Downregulation of H3K27me3 using the small molecule inhibitor EPZ-6438 significantly promoted CRC cell resistance to oxaliplatin. Conversely, inhibition of the activity of demethylase KDM6A/KMD6B by GSK-J4 sensitized CRC to the effect of chemotherapeutic drugs by upregulating H3K27me3 levels in vivo, 1 mm3 tumor sections from PDX#4 and #6 were subcutaneously implanted into nude mice separately. When the tumor size reached 50 ± 10 mm3, we treated the mice with oxaliplatin every 4 days for 4 weeks. The mice bearing tumors from PDX#6 showed increased sensitivity to oxaliplatin than those bearing tumors from PDX#4. The tumor growth rates and weights were significantly inhibited by oxaliplatin treatment in mice bearing tumors from PDX#6, which had high levels of H3K27me3. Likewise, tumors with low H3K27me3 levels (tumors from PDX#4) were slightly inhibited by oxaliplatin . Our results demonstrated that patients with low H3K27me3 level had shorter metastasis-free survival (MFS) times than those with high levels of H3K27me3, which indicated that H3K27 trimethylation might be an independent predictor of stratified risk for MFS . Next, wliplatin . These rBecause of an association between low H3K27me3 level and oxaliplatin resistance, we questioned whether oxaliplatin treatment could induce H3K27 demethylation. First, we assessed the expression of two demethylases, KDM6A and KDM6B, after oxaliplatin treatment in CRC cell lines. Quantitative real-time reverse transcription PCR (qRT-PCR) assay showed a remarkable increase in the mRNA level of KDM6A and KDM6B in HCT116 and SW620 cell lines after oxaliplatin treatment for 24 and 48 h . HoweverDue to the induction of KDM6A and KDM6B by oxaliplatin, we generated KDM6A- and KDM6B-depleted HCT116 and SW620 cells using an shRNA knockdown system . Oxaliplin vitro.Next, we selected two inhibitory molecules that target the enzymes involved in the modification of H3K27 methylation process to further investigate the function of H3K27me3 modification in oxaliplatin-induced apoptosis. GSK-J4 is an inhibitor of KDM6A/KDM6B , and EPZin vivo to GSK-J4 administration. Tumors (1 mm3 sections) from PDX#4 were subcutaneously inoculated into BALB/C nude mice. We subsequently observed tumor growth in the mice following intraperitoneal (i.p.) injection of GSK-J4 combined with oxaliplatin. The results demonstrated that the combination of GSK-J4 and oxaliplatin significantly inhibited the tumor growth rate and weight compared with the group that was treated only with oxaliplatin  assay. The distribution of H3K27me3 was mainly at the sites 1 kb upstream of the NOTCH2 transcription start site (TSS) (p3) and 500 bp upstream of the NOTCH2 TSS (p4) . As an iColon CSCs are thought to be an important cause of chemotherapy resistance , and NOTSince we have demonstrated that H3K27me3 regulates the expression of NOTCH2 and modulates the sensitivity of CRC cells to the chemotherapeutic drug oxaliplatin, we hypothesized that the effect of H3K27me3 modification on drug sensitivity was likely due to its regulation on colorectal CSCs. To characterize the function of H3K27me3 modification in colorectal CSCs, two inhibitors, EPZ-6438 and GSK-J4, were used in an oncosphere assay. The addition of GSK-J4 inhibited oncosphere formation, while EPZ-6438 significantly enhanced this process . Consistin vitro. Next, the combination of GSK-J4 and oxaliplatin significantly inhibited tumor growth compared with that in the group treated only with oxaliplatin in an oxaliplatin-resistant PDX model. Moreover, GSK-J4 remarkably inhibited oncosphere formation and the expression of stemness-related genes. These findings suggest that H3K27me3 might be a potential target for overcoming chemoresistance in CRC.In stem cells, the degree of H3K27me3 is usually low in the promoter region of stem genes. As the degree of H3K27me3 in these promoter regions increases, stem cells differentiate into different types of cells . ClinicaIn this article, we found that overexpression of NOTCH2 does not completely reduce the apoptotic proportion induced by the combination of GSK-J4 and oxaliplatin to the same level as oxaliplatin treatment alone. There might be other genes involved in the process. Our transcriptome sequencing (RNA-seq) analysis showed that GSK-J4 treatment significantly downregulate other stem cell-related genes such as CTNNB1, TEAD4, and TNSK2. However, ChIP assay demonstrated that H3K27me3 cannot be enriched at the promoter regions of these genes (data not shown), which indicated that these genes are not directly regulated by the modification of H3K27me3 on their promoter region. It has been reported that UTX/JMJD3 can enhance the WNT3 gene expression by removing H3K27me3 from its gene promoter, thus promoting β-catenin-mediated gene expression in the process of endoderm differentiation . These rThe effect of H3K27me3 levels on chemoresistance remains controversial in different types of cancers. It has been reported that low EZH2 expression, which reduces H3K27 methylation, promoted drug resistance in BRCA-2-deficient breast cancer cells by the regulation of genomic stability . EPZ-643in vitro . More PDX are still needed to verify the conclusion that we draw. At the same time, Although our results EZH2 is considered to be an oncogene, but inhibition of EZH2 activity in some solid tumors does not inhibit tumor growth due to changes in H3K27 trimethylation caused by EZH2 and also causes changes in H3K27 acetylation . In our in vitro. The combination of GSK-J4 and oxaliplatin predominantly inhibited tumor growth in the oxaliplatin-resistant PDX model. GSK-J4 induced H3K27me3 level and significantly inhibited the self-renewal capability of colorectal CSCs through the downregulation of NOTCH2 expression. Because a crucial role is played by H3K27me3 modification in regulating resistance to oxaliplatin, elevating its expression by GSK-J4 may be an effective strategy to overcome chemoresistance in CRC treatment.In summary, we found that low levels of H3K27me3 were associated with poor prognosis, and increasing H3K27me3 levels sensitized CRC cells to oxaliplatin 3 in size and implanted subcutaneously into the flanks of the nude mice. Drug treatment was initiated when the tumor volume reached 50 ± 10 mm3. Oxaliplatin was administered by i.p. injection (1 mg/kg) every 4 days for 28 days, and GSK-J4 was administered via i.p. injection (100 mg/kg) for 20 consecutive days. After 4 weeks, the mice were sacrificed, and the tumors were dissected. The tumor weight was measured, and the tumor volume was calculated using the following formula: A × B2 × 0.5, with A representing the longest diameter and B the shortest diameter.Four-week-old nude mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences. All experiments were performed under the guidelines for the care and use of laboratory animals. PDX models were established according to previously published reports . The nudIHC analyses were performed as described previously  using anHuman CRC cell lines HCT116 and SW620 were purchased from American Type Culture Collection. Cells were cultured in Dulbecco’s modified Eagle medium containing 10% fetal calf serum medium. All CRC cells were negative for mycoplasma contamination prior to use.Oxaliplatin, GSK-J4, and EPZ-6438 were purchased from Selleck. DAPI (#5748) was obtained from R&D Systems. The following antibodies were used: H3K27me3  for IHC, H3K27me3  for ChIP and western blot, H3K4me3 , H3 , PAPR , γH2A.X , and cleaved caspase 3 .t-test. Primer sequences are presented in Total RNA was isolated using TRIzol reagent (TaKaRa) according to the manufacturer’s protocol. To obtain cDNA, PrimeScript™ RT Master Mix  was used to generate cDNA following the manufacturer’s instructions using 1.5 μg RNA as the template. GAPDH was used as an internal control gene. The qRT-PCR reagents were purchased from TaKaRa, and a 7500 Fast Real-Time PCR System (Applied Biosystems) was used for qRT-PCR. All samples were analyzed in triplicate. The error bars represent the standard error of mean, and statistical significance was calculated using one-tailed, unpaired The harvested cells were washed twice with phosphate buffer saline (PBS), dissolved in loading buffer, and lysed at 100°C for 10 min. Proteins were separated by SDS-PAGE gel and transferred onto a polyvinylidene fluoride (PVDF) membrane . The membrane was blocked in 10% nonfat milk for 30 min followed by immunoblotting with primary and HRP-labeled secondary antibodies.5 cells were plated in 6-well plates for 48 h. After trypsin digestion and washing with PBS, APC-Annexin V (Invitrogen) and 7-AAD (BD Biosciences) were added for 30 min at room temperature in the dark, prior to flow cytometry analysis. The data were analyzed with Kaluza analysis software .A total of 1 × 10In the colony formation assays, cells were cultured continuously in a 10% serum medium for 7 days, then the medium was removed, and the cells were stained with bromophenol blue for 20 min. For sphere formation assays, the cells were suspended in serum-free medium and plated in low-attachment 24-well plates. The serum-free medium contained DMEM/F-12 supplemented with B27 (1:50) supplement, EGF (20 ng/ml), and FGF (10 ng/ml). Seven days after continuous cultivation, the number of spheres was recorded under a microscope.The total mRNA was obtained from HCT116 cells treated with either oxaliplatin or GSK-J4 for 48 h. After total RNA extraction, samples were processed by Hi-Seq 2500 by OE Biotech. Gene expression levels were analyzed by the software HTSeq-count and Cufflinks (version 0.6.1) . All dif6 cells were fixed with formaldehyde for 10 min and sonicated with UCD-300 (Bioruptor). Incubation with antibodies was reversed by cross-linking using the EZ-ChIP immunoprecipitation kit . The antibody used for ChIP was polyclonal rabbit anti-H3K27me3 . Primer sequences (5′–3′) used for PCR amplification are listed in HCT116 cells were treated with oxaliplatin or GSK-J4 for 48 h. Then 1 × 10t-test. Independent prognostic factors were determined by Cox proportional hazard regression models. Differences reached significance at P < 0.01 (**) and P < 0.05 (*).All experiments were performed using three independent replicates from cells. Unless otherwise stated, the data in the graph are presented as mean ± SD, and the statistical significance was evaluated using a two-tailed Student’s Supplementary_material_mjz032Click here for additional data file.