Role of caspases in apoptosis induced by high concentrations of ATG. (A) Effect of zVAD-fmk on DNA fragmentation induced by ATG. The caspase inhibitor zVAD-fmk (100 μM) was added to PBLs for 1 hour, and then cells were incubated with NRS or ATG (250 μg/mL). DNA fragmentation was analyzed at the indicated time by using the F7-26 mAb. The percentage of cells with fragmented DNA is indicated for each histogram. (B) Caspase-3 and -7 processing. PBLs were treated with rabbit ATG at indicated concentrations and lysed at 24 hours, and proteins were separated on SDS-PAGE. (C) Analysis of PARP cleavage. PBLs were incubated with NRS or ATG (250 μg/mL) and preactivated T cells with anti-CD95 (7C11 1 μg/mL). At 24 hours, cells were lysed and proteins were separated on SDS-PAGE. The p85 band corresponds to the caspase-3 cleavage product of PARP. (D) Caspase-9 cleavage. Cells were treated with rabbit ATG and lysed at 24 hours, and proteins were separated on SDS-PAGE and analyzed by Western blotting with an anti-caspase-9 antibody that recognizes the proform (p46) and the cleaved forms (p37/p34) of caspase-9. As positive control, 3-day-activated PBLs were treated with STS (0.5 μM) for 12 hours. Amounts of loaded proteins were controlled for homogeneity by probing membranes with anti-β-actin mAb. All data are from one representative experiment of 2 independent experiments showing similar results.