Figure 5. TRAIL inhibits p21 expression.

(A) C2C12 cells were transduced with lentiviruses expressing shTRAIL or shScramble followed by 2 days of puromycin selection. Cells were then lysed at 0 hr of differentiation followed by western analysis (n=6). (B) C2C12 cells were transduced with lentiviruses expressing shTRAIL-R2 or shScramble followed by 2 days of puromycin selection. Cells were then lysed at 0 hr of differentiation and subjected to western analysis (n=7). (C) Mouse primary myoblasts were transduced with lentiviruses expressing shRNAs overnight, cultured in growth medium for 1 day, and then lysed at 0 hr of differentiation followed by western analysis (n=4). (D) C2C12 cells treated as in (A) were transfected with TRAIL or empty vector, selected with hygromycin and lysed at 0 hr of differentiation, followed by western analysis (n=4). (E) C2C12 cells were induced to differentiate for 6 to 9 hrs, and then treated with vehicle or 100 ng/mL sTRAIL for 15, 30 or 60 min, followed by cell lysis and western analysis (n=5). (F) C2C12 cells were transduced with adenoviruses expressing human full-length TRAIL or luciferase (Control), followed by differentiation for 72 hrs with or without 2 μg/mL Ara-C during the first 24 hr of differentiation. Cells were stained for MHC (green) and DAPI (magenta), and differentiation and fusion indices were quantified (n=3). Expression of TRAIL was confirmed by western blotting. Quantification of p21 protein was performed by densitometry analysis of western blots. The level of p21 protein was determined relative to the level of tubulin, and the data were always normalized to control at each time point. All error bars represent SEM. For (F), one-tailed t-test was performed to compare each data point to control (without Ara-C). For all other panels, two-tailed t-test was performed to compare each data to control (first bar in graph unless otherwise noted). *P < 0.05; **P < 0.01. NS, not significant. Scale bar: 50 μm.