Critical roles of mitochondrial fission and mitochondrial translation in mitochondrial alterations induced by reduced MTF. (A and B) Typical images of dominant-negative mutant of Drp1 (Drp1^K38A)-transfected WT HCT116 cells treated with Acn for 6 h and immunolabeled to detect cytochrome c (green in merged images in A), conjugated Ub (FK2; green in merged images in B), SDHA (red in merged images in A and B), and Tom20 (blue in merged images in A and B). Drp1^K38A-expressing cells are traced with green (A and B) and nuclei (B) with yellow lines. Detail images are from areas outlined with white rectangles. Scale bars represent 20 µm (5 µm in detail images). (C) Quantification of “high”–cytochrome c mitochondria in mock- and Drp1^K38A-transfected WT HCT116 treated with Acn for 6 h. Data represent mean ± SD; n = 3 (n = 100 cells). (D) Quantification of circular mitochondrial ubiquitination in mock- and Drp1^K38A-transfected WT and ATG5^−/− cells treated with Acn for 6 h. Data represent mean ± SD; n = 4; (n = 100 cells). (E–K) WT (E–H) and ATG5^−/− HCT116 cells (I–K) cells were treated with DMSO (control; E and I), Acn (F and J), CP (G), and Acn plus CP (H and K) for 6 h, followed by immunostaining to detect cytochrome c (IMS marker, green), ClpP (matrix marker; red), and Tom20 (OMM marker; blue) and Airyscan image acquisition. Scale bars represent 20 µm (2 µm in detail images).