Effect of B-PAC-1 on procaspase activation and role of caspases in B-APC-1–mediated apoptosis in CLL lymphocytes. Representative immunoblots of executioner procaspase-3 and -7, initiator procaspase-8 and -9, and downstream target PARP protein before and 24 hours after B-PAC-1 treatment (A); quantitation of several immunoblots for these caspases are included as supplemental Figures 2-7. Effect of 24-hour treatment of B-PAC-1 on apoptosis in WT or caspase-8–deficient Jurkat cells (B), in WT, single allele caspase-3 and -7, or caspase-3 and -7 double knockout MEFs (C), and in WT or Bax and Bak double knockout MEF (D). Apoptosis was measured by Annexin V/PI staining assay and unstained cells were considered viable cells; time-matched DMSO-treated cells served as control. Temporal relationship of B-PAC-1–induced reduction in viability (measured by AnnexinV/PI⁻ cells), reduction in mitochondrial outer membrane staining (measured by TMRE staining), and reduction in procaspase-3 protein expression (cleavage in procaspase-3 protein expression measured by immunoblots), after 2 hours to 10 hours of incubation with 10 µM B-PAC-1 (E). Staurosporine, FasL, and PAC-1a were used as positive or negative controls. FasL, Fas ligand; Geomean, geometric mean; STS, Staurosporine.