Figure 3

Treatment with TRAIL/zVAD/CHX or TRAIL/zVAD/HHT does not elicit apoptosis in tumor cells. (A-C) Cells were either left untreated or pretreated with 50 μM zVAD-fmk with or without HHT/CHX (same concentrations as given in Figure  2 ). After one hour, 100 ng/ml of TRAIL were added as indicated. After 24 h of stimulation, complete cell lysates were prepared. Identical amounts of protein (20 μg) were loaded on each lane (from left to right: lane 1–3: negative controls, lane 4–5: necroptosis, lane 6–8: apoptosis as positive control) and PARP-1, cleaved caspase-8 and cleaved caspase-3 were detected in Western blots. (A) The antibody against PARP-1 recognizes the 116-kDa full-length protein and the cleaved 89-kDa form that is only visible in apoptotic lysates (TRAIL, TRAIL/CHX, TRAIL/HHT). (B) The antibody against caspase-8 recognizes the 18-kDa cleaved fragment that is present only in apoptotic lysates (TRAIL, TRAIL/CHX, TRAIL/HHT). Asterisks: non-specific double band that does not match to the size of the 18-kDa cleaved fragment of caspase-8. (C) The antibody against caspase-3 only recognizes the cleaved (active) form of caspase-3 that appears only in apoptotic lysates (TRAIL, TRAIL/CHX, TRAIL/HHT). Detection of actin served as a loading control. (D) Caspase-8 activity was measured in Mz-ChA-1 and HT-29 cells. Extracts were prepared from untreated (●) cells or from cells in which necroptosis (Δ) or apoptosis (□) had been induced as in (A-C), except that pretreatment with zVAD-fmk was for 30 min and that stimulation time was 4 h. As a positive control (×), caspase activity in untreated extracts was stimulated by the addition of dATP and cytochrome c; samples containing only buffer but no extracts served as negative control (○). Caspase-8 activity was determined by measuring the cleavage of a fluorogenic peptide (zIETD-afc) in a time course over 75 minutes as described in Methods.