Mechanism-based combination of B-PAC-1. Effect of B-PAC-1 treatment on IAP expression (A). CLL lymphocytes were treated with or without 10 µM B-PAC-1 for 24 hours and cIAP1 and survivin protein levels were analyzed. Effect of B-PAC-1 treatment on Smac protein expression (B). CLL cells were treated with or without 10 µM B-PAC-1 for 24 hours, and smac protein was immunoblotted. Quantitation of IAP family proteins and smac protein are shown as supplemental Figures 10-11, respectively. Mechanistic combination of B-PAC-1 and Smac-mimetic (Smac066) (C-D). CLL lymphocytes were incubated with B-PAC-1, Smac066, or a combination of both at indicated concentrations for 24 hours. Apoptosis was measured by Annexin V/PI staining assay; time-matched DMSO-treated cells served as control. Data from one patient are shown in panel C. Combination index of B-PAC-1 and Smac066 in 10 patient samples are plotted (D). CLL cells were incubated with indicated concentrations of each drug and their combination for 24 hours, and combination index using the percentage of apoptosis was calculated. CI, combination index (a CI of <1 indicates synergy; calculated using CompuSyn computer software).