Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Histone methyltransferase EZH2 drives podocyte injury and senescence in diabetic nephropathy through STAT3 activation

doi: 10.1007/s00018-026-06136-x

Figure Lengend Snippet: Expression of EZH2 and podocyte injury were enhanced in db/db mice. ( A - B ) Representative images and quantitative analysis of immunohistochemistry staining of EZH2 in the glomeruli of human renal samples (40×). Scale bar = 50 μm. * P < 0.05 vs. control group ( n = 10). ( C ) Correlation analysis between estimated glomerular filtration rate (eGFR) and the expression of EZH2 in DN patients. ( D - E ) Urinary albumin/creatine ratios (uACRs) and blood glucose levels measured in each group. P values vs. db/m group ( n = 6). ( F ) Representative images of periodic acid-Schiff (PAS) staining revealing glomerular alterations in each group (40×). Scale bar = 20 μm. ( G ) Micrographs of podocyte foot processes (green arrows), podocyte foot process effacement and fusion (red arrows) and glomerular basement membrane (GBM, blue arrows) observed by transmission electron microscopy (TEM) in each group. Scale bar = 500 nm. ( H ) Quantification of the number of foot processes in different groups of mice. P values vs. db/m group ( n = 6). ( I - K ) Illustrative confocal microscopic images and quantifications of podocin (green) and B7-1 (red) in each group. Scale bar = 20 μm. P values vs. db/m group ( n = 6). (L-M) Western blotting and quantitative analysis of EZH2, nephrin, podocin, and B7-1 levels in isolated glomeruli in each group. Histone 3 (H3) and β-actin served as loading controls. P values vs. db/m group ( n = 6). CON, control; DN, diabetic nephropathy

Article Snippet: The following antibodies were used for immunofluorescence staining: EZH2 (1:100, 5246 S, CST, USA), pSTAT3 (1:50, 4113 S, CST, USA), podocin (1:200, ab50339, Abcam, UK), WT-1 (1:200, ab89901, Abcam, UK), Flag (1:200, 80010-1-RR, Proteintech, Wuhan, China) and B7-1 (1:200, sc-376012, Santa Cruz Biotechnology, USA).

Techniques: Expressing, Immunohistochemistry, Staining, Control, Filtration, Membrane, Transmission Assay, Electron Microscopy, Western Blot, Isolation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Histone methyltransferase EZH2 drives podocyte injury and senescence in diabetic nephropathy through STAT3 activation

doi: 10.1007/s00018-026-06136-x

Figure Lengend Snippet: Up-regulated EZH2 levels and aggravated podocyte injury in high glucose-cultured MPCs. ( A - B ) Western blotting and quantitative analysis of EZH2, synaptopodin, podocin, and B7-1 levels in mouse podocyte cells (MPCs). H3 and β-actin served as loading controls. ns: P > 0.05 vs. NG group. P values vs. NG group ( n = 3). ( C - D ) Illustrative confocal microscopic images and quantification showed EZH2 (green) in the MPCs counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar = 20 μm. ns: P > 0.05 vs. NG group. P values vs. NG group ( n = 3). NG, normal glucose; HM, high mannitol; HG, high glucose

Article Snippet: The following antibodies were used for immunofluorescence staining: EZH2 (1:100, 5246 S, CST, USA), pSTAT3 (1:50, 4113 S, CST, USA), podocin (1:200, ab50339, Abcam, UK), WT-1 (1:200, ab89901, Abcam, UK), Flag (1:200, 80010-1-RR, Proteintech, Wuhan, China) and B7-1 (1:200, sc-376012, Santa Cruz Biotechnology, USA).

Techniques: Cell Culture, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Histone methyltransferase EZH2 drives podocyte injury and senescence in diabetic nephropathy through STAT3 activation

doi: 10.1007/s00018-026-06136-x

Figure Lengend Snippet: The EZH2 inhibitor GSK126 alleviated podocyte injury and glomerular senescence in db/db mice. ( A - B ) uACRs and blood glucose levels measured in each group. ns: P > 0.05 vs. db/db group. P values vs. db/db group ( n = 6). ( C ) Representative images of PAS staining of glomeruli in each group (40×). Scale bar = 20 μm. ( D ) Micrographs of podocyte foot processes (green arrows), podocyte foot process effacement and fusion (red arrows) and GBM (blue arrows) observed by TEM in each group. Scale bar = 500 nm. ( E - F ) Western blotting and quantitative analysis of EZH2, H3K27me3, nephrin, podocin, B7-1, p21, and Klotho in each group. H3 and β-actin served as loading controls. ns: P > 0.05 vs. db/db group. P values vs. db/db group ( n = 6). ( G ) Illustrative confocal microscopic images of podocin (green) and B7-1 (red) in different groups. Scale bar = 20 μm. ( H - I ) Graphs of SA-β-gal (yellow arrows) staining and immunohistochemistry staining for p16 INK4A in each group (40×). Scale bar = 20 μm. The dashed lines represent the enlarged images of the area indicated by the arrows. ( J - K ) Quantifications of the number of foot processes and p16 INK4A in different groups of mice. ns: P > 0.05 vs. db/db group. P values vs. db/db group ( n = 6). ( L - M ) Quantitative analysis of podocin and B7-1 in the glomeruli of different groups. ns: P > 0.05 vs. db/db group. P values vs. db/db group ( n = 6). β-CD, SBE-β-CD; GSK126, GSK2816126A

Article Snippet: The following antibodies were used for immunofluorescence staining: EZH2 (1:100, 5246 S, CST, USA), pSTAT3 (1:50, 4113 S, CST, USA), podocin (1:200, ab50339, Abcam, UK), WT-1 (1:200, ab89901, Abcam, UK), Flag (1:200, 80010-1-RR, Proteintech, Wuhan, China) and B7-1 (1:200, sc-376012, Santa Cruz Biotechnology, USA).

Techniques: Staining, Western Blot, Immunohistochemistry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Histone methyltransferase EZH2 drives podocyte injury and senescence in diabetic nephropathy through STAT3 activation

doi: 10.1007/s00018-026-06136-x

Figure Lengend Snippet: EZH2 methylates and activates STAT3 in podocytes. ( A - B ) Western blotting and quantitative analysis of pSTAT3/STAT3 in each group. P values vs. db/m group ( n = 6). ( C - D ) Western blotting and quantitative analysis of pSTAT3/STAT3 in MPCs. ns: P > 0.05 vs. NG group. P values vs. NG group ( n = 3). ( E - F ) Western blotting and quantifications of pSTAT3/STAT3 in different groups. ns: P > 0.05 vs. db/db group. P values vs. db/db group ( n = 6). ( G - H ) Western blotting and quantitative analysis of pSTAT3/STAT3 and H3K27me3 in MPCs. ns: P > 0.05 vs. HG group. P values vs. HG group ( n = 3). ( I ) Co-immunoprecipitation (Co-IP) revealed a relationship between EZH2 and STAT3, as well as EZH2 and pSTAT3. The Input group served as a positive control, and the IgG group served as a negative control. ( J ) Illustrative confocal microscopic images of EZH2 (red) and pSTAT3 (green) in MPCs counterstained with DAPI (blue). Scale bar = 20 μm. ( K ) Image of molecular docking between EZH2 (pink) and STAT3 (green). ( L ) Co-IP showed the interaction between EZH2 and STAT3 after EZH2 inhibition by GSK126. ( M ) The methylation status of lysine residues of STAT3 was evaluated by Co-IP in MPCs treated with NG or HG. ( N ) The methylation status of lysine residues of STAT3 after EZH2 knockdown (KD) by adenovirus

Article Snippet: The following antibodies were used for immunofluorescence staining: EZH2 (1:100, 5246 S, CST, USA), pSTAT3 (1:50, 4113 S, CST, USA), podocin (1:200, ab50339, Abcam, UK), WT-1 (1:200, ab89901, Abcam, UK), Flag (1:200, 80010-1-RR, Proteintech, Wuhan, China) and B7-1 (1:200, sc-376012, Santa Cruz Biotechnology, USA).

Techniques: Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Positive Control, Negative Control, Inhibition, Methylation, Knockdown

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Histone methyltransferase EZH2 drives podocyte injury and senescence in diabetic nephropathy through STAT3 activation

doi: 10.1007/s00018-026-06136-x

Figure Lengend Snippet: Overexpression of STAT3 eliminated the reversal role of EZH2 inhibition against podocyte damage and senescence in db/db mice. ( A ) Flow chart of treatment, sampling and observation end points for the db/db mice. ( B ) Illustrative immunofluorescence staining images of Flag (red) and Wilms Tumor-1 (green) in db/db + EZH2 KD + STAT3 OE group mice. Scale bar = 20 μm. ( C - D ) uACRs and blood glucose levels were measured in different groups ( n = 6). ( E ) Representative images of PAS staining of glomeruli in each group (40×). Scale bar = 20 μm. ( F ) Micrographs of podocyte foot processes (green arrows), podocyte foot process effacement and fusion (red arrows) and GBM (blue arrows) as observed by TEM in each group. Scale bar = 500 nm. ( G - H ) Western blotting and quantitative analysis of p21, Klotho, nephrin, podocin, and desmin levels in each group. H3 and β-actin served as loading controls. P values vs. db/db + EZH2 KD group ( n = 6). ( I ) Illustrative confocal microscopic images of podocin (green) and B7-1 (red) in each group. Scale bar = 20 μm. ( J - K ) Graphs of SA-β-gal (yellow arrows) staining and immunohistochemistry staining for p16 INK4A in each group (40×). Scale bar = 20 μm. The dashed lines represent the enlarged images of the area indicated by the arrows. ( L - O ) Quantifications of the number of foot processes, podocin, B7-1 and p16 INK4A in different groups of mice ( n = 6). OE, overexpression

Article Snippet: The following antibodies were used for immunofluorescence staining: EZH2 (1:100, 5246 S, CST, USA), pSTAT3 (1:50, 4113 S, CST, USA), podocin (1:200, ab50339, Abcam, UK), WT-1 (1:200, ab89901, Abcam, UK), Flag (1:200, 80010-1-RR, Proteintech, Wuhan, China) and B7-1 (1:200, sc-376012, Santa Cruz Biotechnology, USA).

Techniques: Over Expression, Inhibition, Sampling, Immunofluorescence, Staining, Wilms Tumor Assay, Western Blot, Immunohistochemistry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Histone methyltransferase EZH2 drives podocyte injury and senescence in diabetic nephropathy through STAT3 activation

doi: 10.1007/s00018-026-06136-x

Figure Lengend Snippet: STAT3 overexpression counteracts podocyte protection mediated by EZH2 inhibition in high glucose-treated MPCs. ( A - B ) Western blotting and quantitative analysis of p21, Klotho, synapotopodin, podocin, and B7-1 levels. H3 and β-actin served as loading controls. P values vs. HG + EZH2 KD group ( n = 3). ( C - D ) Graphs and quantitative analysis of SA-β-gal (yellow arrows) staining in MPCs ( n = 3). ( E - F ) Representative images of western blotting and quantifications of p21, Klotho, synapotopodin, podocin, and B7-1 levels. H3 and β-actin served as loading controls. P values vs. HG + EZH2 KD group ( n = 3)

Article Snippet: The following antibodies were used for immunofluorescence staining: EZH2 (1:100, 5246 S, CST, USA), pSTAT3 (1:50, 4113 S, CST, USA), podocin (1:200, ab50339, Abcam, UK), WT-1 (1:200, ab89901, Abcam, UK), Flag (1:200, 80010-1-RR, Proteintech, Wuhan, China) and B7-1 (1:200, sc-376012, Santa Cruz Biotechnology, USA).

Techniques: Over Expression, Inhibition, Western Blot, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Histone methyltransferase EZH2 drives podocyte injury and senescence in diabetic nephropathy through STAT3 activation

doi: 10.1007/s00018-026-06136-x

Figure Lengend Snippet: A schematic diagram illustrates the multifaceted role of the EZH2 in podocyte senescence upon diabetic insult and the protective effect of targeting EZH2. The present study demonstrated that EZH2 is highly expressed in DN and negatively correlated with eGFR. EZH2-mediated STAT3 activation promotes podocyte senescence. The aforementioned pathogenic pathways contribute to kidney aging in DN and can be mitigated by targeting EZH2, either genetically through adeno-associated virus-mediated knockdown, or pharmacologically using small-molecule inhibitors such as GSK126

Article Snippet: The following antibodies were used for immunofluorescence staining: EZH2 (1:100, 5246 S, CST, USA), pSTAT3 (1:50, 4113 S, CST, USA), podocin (1:200, ab50339, Abcam, UK), WT-1 (1:200, ab89901, Abcam, UK), Flag (1:200, 80010-1-RR, Proteintech, Wuhan, China) and B7-1 (1:200, sc-376012, Santa Cruz Biotechnology, USA).

Techniques: Activation Assay, Virus, Knockdown