Mechanism of B-PAC-1–mediated apoptosis. Role of exogenous zinc ions in B-PAC-1–mediated apoptosis in CLL lymphocytes (A). Cells were treated with 100 µM exogenous zinc ions in the presence or absence of PAC-1a, ABT199, STS, or B-PAC-1 at indicated concentrations for 24 hours. Titration of zinc concentrations (B). ZnSO₄ at 10 µM, 50 µM, and 100 µM was added to CLL lymphocytes in the presence of B-PAC-1 at 0 hours (□) or after 5 hours of B-PAC-1 incubation (▪). Role of caspase inhibitors in B-PAC-1–induced apoptosis (C-D). Cells were treated with 50 µM Z-VAD or Q-VD (pan-caspase inhibitors) in the presence or absence of STS or B-PAC-1 for 24 hours and the percentage of apoptosis was measured (C). Representative immunoblot of samples (n = 2) treated with Z-VAD and Q-VD for 24 hours in the presence and absence of B-PAC-1 and ABT-199 (a Bcl-2 antagonist as a positive control) and 0-hour sample (right after isolation of CLL lymphocytes from blood) (D). Reversibility of B-PAC-1–induced apoptosis (E). CLL lymphocytes were incubated with PAC-1a or B-PAC-1 and either not washed or washed after 5 hours (E) and viability (% AnnexinV/PI⁻ cells) was measured. Working model of B-PAC-1 mechanism of action (F). PAC-1a, negative control.