Loss of Bad does not affect mitochondrial release of cytochrome c, caspase processing or loss of mitochondrial membrane potential following IL-3 withdrawal. (A) Cytochrome c release from mitochondria was examined 48 hours following IL-3 withdrawal. Cells were cultured in the presence or absence of IL-3 for 48 hours, then permeabilized, fixed, and stained with antibodies to cytochrome c prior to analysis by flow cytometry to determine the percentage of cells that had lost mitochondrial cytochrome c. Data show the mean ± SEM of 3 independent experiments using 2 independent lines of each genotype. (B) Caspase inhibition diminishes the percentage of cells that are stained by both annexin V and PI following IL-3 withdrawal. Cells were cultured for 24 hours in the presence or absence of IL-3 with or without the broad-spectrum caspase inhibitor Q-VD-Z-OPh (100 µM). Cell viability was determined by annexin V-FITC staining and PI uptake. Columns show the mean ± SEM of 3 independent experiments using 2 independent lines of each genotype. (C) Western blots of lysates from cells cultured for 24 hours with IL-3 (lanes 1, 4, 7, 10, 13, and 16), 24 hours without IL-3 (lanes 2, 5, 8, 11, 14, and 17), and 48 hours without IL-3 (lanes 3, 6, 9, 12, 15, and 18) were probed with an antibody to caspase-3. Arrows show full-length and processed caspase-3, respectively. Probing with an antibody to β-actin was used as a loading control. (D) Changes in mitochondrial membrane potential following IL-3 withdrawal. Cells were cultured without IL-3 for the indicated lengths of time, stained with DIOC₆⁽³⁾, and analyzed by flow cytometry to determine the percentage of cells that had lost their mitochondrial membrane potential. Dead or dying cells have diminished DIOC₆⁽³⁾ fluorescence, which is detected in the FL1 channel. The results show the mean ± SEM of 3 independent experiments, each using 2 independent lines of each genotype. (E) Histograms of DIOC₆⁽³⁾ fluorescence following IL-3 withdrawal and after restoration of IL-3. At each time during factor starvation and following readdition of IL-3 (indicated at the left of each panel), cells were harvested and stained with DIOC₆⁽³⁾. The histograms shown are from a single experiment representative of 3 independent analyses.