Recruitment of autophagy proteins to the ubiquitinated IMM in Acn-treated cells. (A–G) WT (A, B, D, F, and G) and ATG5^−/− (C, E, and H) HCT116 cells were treated with Acn (A, C, E, F, and H) or cotreated with Acn and BafA1 (B, D, and G). Cells were labeled with anti-Ub FK2 mAb (blue in A–H), anti-Tom20 antibody (red in A–H), anti-p62 antibody (green in A–C), anti-OPT antibody (OPT; green in D and E), and anti-LC3 antibody (green in F–H). Two typical examples of mitochondria with OMM severing are shown for each condition. Scale bars represent 1 µm. (I) The localization of p62 and LC3 with circular Ub in cells treated and exemplified in A–C and F–H was blindly quantified. n = 3 (n > 50 per experiment). (J) Quantification of positions of p62, OPT, and LC3 relative to Ub signal. The insert shows mean ± SD of data shown in the main graph. n = 50 per condition. Data pulled from n = 3 independent experiments. Examples of fluorescence intensity profiles used for quantifications are shown on the right of images in A and B. (K) p62-positive mitochondria are deficient in cytochrome c. Cells treated with Acn for 6 h were immunostained to detect Tom20 (red), cytochrome c (cyt. c; blue), and p62 (green). Typical examples are shown. Arrowheads point to p62-positive/cytochrome c–negative mitochondria. 100% of p62-positive mitochondria are deficient in cytochrome c signal (n = 2, n = 50 per experiment). Scale bars represent 1 µm.