Figure 3

Both p53 and caspase-8 functions are needed for etoposide-induced caspase activation in HOC313 cells. A, effects of temperature downshift on the cleavage of the procaspases and PARP in drug-treated HOC313 cells and HOC313/c8_3 cells. The cells treated with drugs as in Figure 2B were subjected to Western blotting for PARP, and the caspases-8, -9 and -3. β-actin was used as a loading control. B, effects of temperature downshift on caspase 3/7 activities in drug-treated HOC313 cells and HOC313/c8_3 cells. The cells treated with drugs as in Figure 2B for 12 h were subjected to Caspase-Glo 3/7 assay. Results are expressed as fold induction compared with untreated control. The bars show the SD. The asterisk indicates a significant difference (p < 0.05) between 32.5°C and 37°C in etoposide-treated HOC313/c8_3 cells (t-test). C, the caspase-8 (C360S) mutant did not induced apoptosis in etoposide-treated HOC313 cells at 32.5°C. HOC313 cells were transiently transfected with the indicated expression vectors by electroporation. Twenty-four hours after transfection, cells were treated with etoposide (100 μg/ml) at 32.5°C. DNA profiles were analyzed 40 h after drug treatment as in Figure 2B (left panel). Caspase-8 expression was analyzed by Western blot using anti-FLAG antibody 24 h after drug treatment (right panel).