Fig. 6

Effects of ceramide and p21 knockdown on the cell cycle and survival of senescent myoblasts. A Cdkn1a silencing significantly increased the viability of ceramide-treated myoblasts (n = 5). B Cell cycle analysis showed that ceramide-induced G1 cell cycle arrest was attenuated by Cdkn1a silencing (n = 5). *p < 0.05, **p < 0.01, ^###p < 0.001 by unpaired t-test. C FACS analysis based on caspase-3/7 activity following ceramide treatment, with or without Cdkn1a silencing, confirmed increased caspase-3/7 activity with Cdkn1a knockdown. D Cell sorting based on caspase-3/7 activity following ceramide treatment and Cdkn1a silencing showed results consistent with (E) TUNEL staining (n = 3). Compared with cells with low caspase-3/7 activity, cells with high caspase-3/7 activity exhibited a greater number of (F) SA-β-gal-positive (n = 11) and (G) γH2AX-positive senescent cells (n = 3). Representative images acquired using a microscope at 10× magnification are shown. F Asterisks indicate senescent myoblasts. *p < 0.05, ***p < 0.001 by unpaired t-test. H BrdU assay shows that siCDK restores the proliferation capacity of ceramide-treated myoblasts (n = 6 per group). Vertical bars indicate standard errors. Two-way analysis of variance (ANOVA) revealed a significant time × group interaction (F = 10.88, p < 0.001). **p < 0.01, ***p < 0.001 (siCON versus siCON + ceramide group [siCON + Cera]); ^##p < 0.01, ^###p < 0.001 (siCON + Cera versus siCDK + ceramide [siCDK + Cera]) by unpaired t-test. I Ki67 staining revealed that ceramide treatment inhibits the cell cycle, which is restored by Cdkn1a silencing (n = 3). Representative images acquired using a microscope at 10× magnification are shown. Representative images from triplicate experiments are shown. γH2AX, H2A histone family member X; Ki67, antigen Kiel 67; DAPI, 4′,6-diamidino-2-phenylindole