XIAP inhibition promotes Bcl-2 cleavage and Bak conformational change in Bcl-2–overexpressing Jurkat cells. (A) Jurkat leukemia cells transfected with Bcl-2 were treated for 12 hours with 1.6 ng/mL TRAIL and/or 10 nM XIAP inhibitor 2, control compound, or DMSO. Cleavage of Bcl-2 and caspase-3 was analyzed by Western blotting. Arrowheads indicate cleavage fragments. (B) Jurkat leukemia cells transfected with Bcl-2 were treated for 12 hours with 0.4 ng/mL TRAIL and/or 10 nM XIAP inhibitor 2, control compound, or DMSO. Bak conformational change was assessed by FACS analysis. Fluorescence intensity (x-axis) is plotted against cell counts (y-axis). A representative experiment of 3 experiments is shown (thin line indicates untreated cells; solid line, TRAIL-treated cells). (C,D) Jurkat leukemia cells transfected with Bcl-2 were treated for 48 hours with 0.4 ng/mL TRAIL and/or 10 nM XIAP inhibitor 2, control compound, or DMSO in the absence (top panels) or presence (bottom panels) of 20 μM zDEVD.fmk. Mitochondrial transmembrane potential (C) and cytochrome c release (D) were assessed by FACS analysis. Mean and SD of 3 experiments each performed in triplicate are shown. For statistical analysis, t test was performed comparing samples treated with XIAP inhibitor 2 and TRAIL in the absence (top panels of C and D) and presence (bottom panels of C and D) of zDEVD.fmk. #P < .05; *P < .01.