Figure 3. H2O2 induces FOXO4 nuclear translocation and cellular senescence in TM3 Leydig cells. (A) SA-β-gal assay showing TM3 Leydig cells with increased SA-β-gal activity after 48 h exposure to 100 μΜ H2O2 in serum-free medium. Scale bar: 100 μm. (B) Immunofluorescent staining showing that H2O2-induced senescent TM3 Leydig cells express FOXO4 predominantly in the nucleus, while controls express FOXO4 in the cytoplasm. Scale bar: 50 μm. (C–E) Western blots of separating nuclear and cytoplasmic extracts showing a significant increase in FOXO4 expression in H2O2-induced senescent TM3 Leydig cells, and FOXO4 concentrated in the nucleus. (F–I) Western blots of total protein revealing the levels of p53, Ser15-phopho-p53 and p21 are significantly elevated in H2O2-induced senescent TM3 Leydig cells. (J–L) Western blots revealing that levels of the testosterone synthesis-related proteins CYP11A1 and CYP17A1 are significantly decreased in H2O2-induced senescent TM3 Leydig cells. Data presented are representative of three independent experiments. Data depict the mean ± SD. *P<0.05.