Cytochrome c, Bax, and apoptosis in Acn- and ABT-737-treated cells. (A and B) WT HCT116 cells treated with Acn (A) or ABT-737 (B) for 6 h were immunostained to detect cytochrome c (blue), Tom 20 (red) and Bax (green), followed by Airyscan imaging. Consistent data were obtained in n = 3 independent experiments. Scale bars represent 10 µm, and 1.0 µm in detail images. (C and D) Imaging of cytochrome c in apoptotic cells. To induce apoptotic OMM permeabilization WT (C) and Bax^−/−/Bak^−/− DKO (D) cells were treated with ABT-737 for 6 h. Cells were immunostained to detect cytochrome c (green) and Tom20 (red). Scale bars represent 10 µm. (E) Number of cells showing cytosolic cytochrome c (as in cells traced with blue lines in C) were quantified. Data represent mean ± SD; n = 150. (F) Western blot analyses of caspase 9 and PARP in WT and ATG5^−/− HCT116 cells treated as indicated in the figure. Asterisks indicate nonspecific proteins detected by anti-caspase 9 and anti-caspase 1 antibodies. Caspase 1, which was not detectably processed, was used as a loading control.