ABT-737 plus L-ASP caused enhanced apoptosis, mitochondrial membrane depolarization, and release of mitochondrial cytochrome c. Annexin V–FITC and JC-1 assay. COG-LL-317 cells were incubated with ABT-737 (2.5 μM), L-ASP (2.5 IU/mL), or the combination for 6 hours. Then, cells were incubated with annexin V–FITC and PI (A) or JC-1 (B,C) and analyzed by flow cytometry. (A) Bars show the percentage of cells that were annexin V–FITC⁺/PI−, defined as apoptotic, and error bars represent standard deviation. (B) The loss of Δψm by ABT-737, L-ASP, or ABT-737 + L-ASP was measured by flow cytometry using the JC-1 mitochondrial probe. The bars show the percentage of mitochondrial membrane–depolarized cells. (C) Cytograms showing representative JC-1 assays for mitochondrial membrane potential. The transition of red fluorescence to green indicates mitochondrial membrane depolarization by the drug(s). The values represent the percentage of mitochondrial membrane-depolarized cells. Total number of events analyzed for each condition was 10 000. (D) Cytochrome c release after drug treatment. Cytosolic extracts from COG-LL-317 that had been incubated for 6 hours with ABT-737 (2.5 μM), L-ASP (2.5 IU/mL), or both drugs in combination were prepared and immunoblotted with cytochrome c antibody. The blot shown is representative of 2 separate experiments.