CTLs activate the mitochondrial apoptotic pathway in Bcl-2–overexpressing cells. (A) CTLs induce mitochondrial electrochemical potential loss in target cells overexpressing Bcl-2. Jneo cells (top row) and JBcl-2 cells (bottom row) were preloaded with CTFR (FL-4) prior to incubation with CTLs (E/T ratio = 2:1) or grB/AD (20 nM grB). Cells were stained with the mitochondrial potential indicator dye TMRE, and FL-4⁺ target cells were analyzed for TMRE loss in the FL-2 channel. This is a representative example of 1 of 4 independent experiments done in triplicate. (B) CTLs induce mitochondrial electrochemical potential loss in target cells overexpressing Bcl-2. Jneo cells (□) and JBcl-2 cells (■) were preloaded with CTFR (FL-4) and incubated for 2 hours with CTLs at E/T ratios of 0.25:1, 0.5:1, 1:1, and 2:1 (CTLs), or 2.5 nM, 5 nM, 10 nM, and 20 nM grB with AD, then loaded with the mitochondrial potential indicator dye TMRE. FL-4⁺ target cells were analyzed for TMRE loss by flow cytometry. The numbers indicate the average of 4 independent experiments done in triplicate, with the corresponding standard deviation. (C) CTLs induce MOMP in target cells overexpressing Bcl-2. Jneo cells (□) and JBcl-2 cells (■) were preloaded with CTFR (FL-4) and incubated for 2 hours with CTLs at an E/T ratio of 2:1 or grB/AD (10 nM grB). Cells were gently permeabilized with digitonin to release soluble cytosolic proteins prior to fixation. Cells were then completely permeabilized with saponin and processed for intracellular staining for Smac/DIABLO (left panel) or COXIV (right panel).