Figure 5.

Phospholipase activity mediates the mitochondrial pathway of TRAIL-induced apoptosis. A, TRAIL induces caspase-8 processing in a time-dependent manner: HeLa cells were subjected to TRAIL (10 ng/mL) treatment for the indicated times and their lysates were immunoblotted for caspase-8 (p20 antibody, Santa Cruz Biotechnology) and actin as loading control. Bottom, densitometric analysis of the p20 band data from three independent experiments. B and C, phospholipase activity does not block Bid and caspase-8 processing but affects cytochrome c release and caspase-9 processing: HeLa cells were incubated first with increasing concentrations of D609 for 30 minutes and then treated with TRAIL (10 ng/mL) for 6 hours. Whole cell lysates were immunoblotted for caspase-8, Bid, caspase-9, and caspase-3, whereas cytosolic fractions were blotted for cytochrome C; protein loading was evaluated with actin re-blots (bottom). Densitometric analysis of the bands for p10 and cytochrome C was done using data from three independent experiments (C, bottom). **, 0.05 < P < 0.10, statistically significant two-sided Wilcoxon test compared with TRAIL alone treatment. D, z-VAD is more potent than D609 in inhibiting TRAIL-induced caspase-8 processing: Lysates of HeLa cells were preincubated with 100 μmol/L z-VAD or 50 Amol/L D609 for 30 minutes as indicated and then treated with TRAIL (10 ng/mL). Densitometric analysis of the p20 band was done using data from three independent experiments (bottom). **, 0.05 < P < 0.10, statistically significant two-sided Wilcoxon test compared with TRAIL treatment alone.