Fig. 3.

Enhancer of zeste homolog 2 (EZH2) inhibition attenuated high glucose (HG)-induced fibroblast differentiation and extracellular matrix (ECM) protein synthesis. (A) Western blot analysis of the expression of phospho-EZH2 (p-EZH2), EZH2, and histone H3 lysine 27 trimethylation (H3K27me3) in cardiac fibroblasts (CFs) after HG and GSK126 (500 nM) treatment (n=3 in each group). (B) Inhibition of EZH2 with GSK126 in CFs and Western blot analysis of protein levels of ECM-related genes and α-smooth muscle actin (α-SMA; n=3 in each group). (C) Representative images of immunofluorescence images of α-SMA expression in CFs with different treatment (red, α-SMA; blue, 4´,6-diamidino-2-phenylindole [DAPI]; scale bar 50 μm). (D) Inhibition of EZH2 with GSK126 under HG treatment for 72 hours, transwell assay was performed (scale bar 200 μm, n=3 in each group). (E) Inhibiting of EZH2 with GSK126 under HG treatment for 72 hours, scratching tests was performed (scale bar 400 μm, n=8 in each group). (F) CFs proliferation were measured with cell counting kit 8 (CCK-8) under HG treatment of for 72 hours with/without GSK126 (n=3 in each group). Data are presented as mean±standard error of the mean. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TGF-β, transforming growth factor-β; NG, normal glucose; DMSO, dimethyl sulfoxide; OD, optical density. ^aP< 0.05, ^bP<0.01.