Figure 4
p73 is responsible for etoposide-induced caspase-8 activation and apoptosis in drug-sensitive Ca9-22 cells. A, the induction of endogenous p73 following drug treatment. Cells treated with the drugs as in Figure 2B were subjected to Western blotting with p73, caspase-8 or caspase-9 antibodies. B, C and D, Ca9-22 cells were transfected with either p73 or control siRNA. At 48 h after siRNA transfection, cells were treated with cisplatin (5 μg/ml) or etoposide (50 μg/ml). DNA profiles were analyzed by LSC 30 h after drug treatment (B, upper panel). The horizontal and vertical axes represent the DNA content and cell number, respectively. Apoptotic cell population is indicated as a percentage of the sub-G1 fraction. p73 expression was analyzed by Western blot 30 h after drug treatment (B, lower panel). Caspase 3/7 activity was analyzed at the indicated time after drug treatment (C). Results are expressed as fold induction compared with untreated control. The bars represent the SD. The asterisk indicates a significant difference (p < 0.05) between p73 siRNA-1 and control siRNA treatment cells (t-test). The cleavage of procaspases was analyzed by western blot with caspases-3 and -8 antibodies 24 h after drug treatment (D). E and F, Ca9-22 cells were transfected with either caspase-8 or control siRNA. At 48 h after siRNA transfection, cells were treated with etoposide (100 μg/ml). DNA profiles were analyzed by LSC 24 h after drug treatment as in B (E). The expression and cleavage of procaspases and Bid was analyzed by western blot with caspases-3, -8, -9, and Bid antibodies 24 h after drug treatment (F). β-actin was used as a loading control.