Induction of mosaic cytochrome c distribution in neuroblastoma, HeLa cells, and Bax/Bak DKO HCT116 cells. (A–D) M17 neuroblastoma (A and B) and HeLa (C and D) cells were treated with DMSO (control; A and C) or Acn (B and D) for 6 h, followed by immunostaining to detect cytochrome c (green in A–D), ClpP (red in A and B), and Tom20 (red in C and D) and Airyscan image acquisition. Detail images are from areas outlined with light blue rectangles. Scale bars represent 20 µm (5 µm in detail images). (E–I) Depletion of Bax and Bak does not affect Acn-induced mosaic distribution of cytochrome c, OMM severing and IMM ubiquitination. Bax^−/−/Bak^−/− DKO HCT116 cells were treated with DMSO (vehicle; E) or Acn (F) for 6 h, followed by immunostaining to detect cytochrome c (green), ClpP (red), and Tom20 (blue) and Airyscan image acquisition. Arrowheads in F (detail) point to the high–cytochrome c mitochondria. Scale bars represent 20 µm (2 µm in detail images). (G) Bax^−/−/Bak^−/− DKO cells treated with Acn for 6 h were immunostained to detect Ub (anti-Ub FK2 mAb; light blue) and Tom20 (red) followed by image acquisition. High magnification of areas marked with rectangles are shown in “detail” images a and b. Scale bars represent 10 µm (1 µm in detail images; a and b). (H) Quantification of circular Ub FK2-positive structures in control and Acn-treated WT and Bax^−/−/Bak^−/− DKO cells. Data represent mean ± SD; n = 50 cells per condition. Similar results were obtained in n = 3 independent experiments. **, P < 0.01; ns indicates nonsignificant; one-way ANOVA with Bonferroni correction (α = 0.05). (I) Western blot analysis of expression levels of Bax and Bak in WT and Bax^−/−/Bak^−/− DKO cells. Tom20 was used as a loading control.