Figure S6.
OMM severing, IMM ubiquitination and distribution of TRAP1 and CPOX in Acn-treated HCT116 and HeLa cells.(A–C) Examples of mitochondria with severed OMM from WT HCT116 (A and B) and WT HeLa (C) cells treated with Acn for 6 h and immunostained to detect conjugated Ub (FK2; green in A–C), Tom20 (blue in A–C), Tom40 (red in A), Fis1 (red in B), and SDHA (red in C). Scale bars represent 1 µm. (D) Quantification of circular mitochondrial ubiquitination in WT HeLa cells treated as indicated in the figure. Data are represented by mean ± SD; n = 3 (n = 60 cells per experiment). *, P < 0.05 versus control; ns indicates nonsignificant; Kruskal-Wallis with Dunn post-hoc analysis (α = 0.05). (E) Total cell lysates obtained from HeLa (cervical adenocarcinoma), HCT116 (colorectal carcinoma), H4 (neuroglioma), HepG2 (hepatocellular carcinoma), SH-SY5Y (neuroblastoma), U251 (glioblastoma), A549 (lung epithelial carcinoma), and HUH7 (hepatocellular carcinoma) were subjected to Western blot to detect Parkin. The upper “Parkin” panel show blots detected with a standard ECL detection reagent (Pico), and the Parkin panel second from the top was detected using a more sensitive ECL detection reagent (Femto). Rat brain lysate was included as a reference. Tom20 was used as loading control. Numbers in the bottom panel indicate expression of Parkin in all analyzed cells relative to SH-SY5Y neuroblastoma cells that showed the highest expression of this protein. The values were normalized to Tom20 levels. (F–K) Distribution and expression of TRAP1 and CPOX was analyzed in WT and Drp1−/− HCT116 and WT HeLa cells. WT (F) and Drp1−/− HCT116 (G and H) cells and WT HeLa cells (I and J) were treated with DMSO (G and I) or Acn (F, H, and J) for 6 h followed by immunostaining to detect cytochrome c (Cyt. c; green), Tom20 (blue), TRAP1 (red in F), and CPOX (red in G and H). Detail images are from areas marked with white rectangles. Scale bars represent 20 µm in F–J; 2 µm in detail images in F, G, and H; and 5 µm in detail images in I and J. Arrowheads in F indicate mitochondria with high cytochrome c/low TRAP1 (red), high TRAP1/low cytochrome c (yellow), and high cytochrome c/high TRAP1 (blue). (K) Expression levels of CPOX, TRAP1, and cytochrome c in WT HeLa cells and WT, ATG5−/−, and Drp1−/− HCT116 cells was analyzed by Western blot. Tom20 was used as a loading control. Two exposures of CPOX blot are shown.

OMM severing, IMM ubiquitination and distribution of TRAP1 and CPOX in Acn-treated HCT116 and HeLa cells. (A–C) Examples of mitochondria with severed OMM from WT HCT116 (A and B) and WT HeLa (C) cells treated with Acn for 6 h and immunostained to detect conjugated Ub (FK2; green in A–C), Tom20 (blue in A–C), Tom40 (red in A), Fis1 (red in B), and SDHA (red in C). Scale bars represent 1 µm. (D) Quantification of circular mitochondrial ubiquitination in WT HeLa cells treated as indicated in the figure. Data are represented by mean ± SD; n = 3 (n = 60 cells per experiment). *, P < 0.05 versus control; ns indicates nonsignificant; Kruskal-Wallis with Dunn post-hoc analysis (α = 0.05). (E) Total cell lysates obtained from HeLa (cervical adenocarcinoma), HCT116 (colorectal carcinoma), H4 (neuroglioma), HepG2 (hepatocellular carcinoma), SH-SY5Y (neuroblastoma), U251 (glioblastoma), A549 (lung epithelial carcinoma), and HUH7 (hepatocellular carcinoma) were subjected to Western blot to detect Parkin. The upper “Parkin” panel show blots detected with a standard ECL detection reagent (Pico), and the Parkin panel second from the top was detected using a more sensitive ECL detection reagent (Femto). Rat brain lysate was included as a reference. Tom20 was used as loading control. Numbers in the bottom panel indicate expression of Parkin in all analyzed cells relative to SH-SY5Y neuroblastoma cells that showed the highest expression of this protein. The values were normalized to Tom20 levels. (F–K) Distribution and expression of TRAP1 and CPOX was analyzed in WT and Drp1−/− HCT116 and WT HeLa cells. WT (F) and Drp1−/− HCT116 (G and H) cells and WT HeLa cells (I and J) were treated with DMSO (G and I) or Acn (F, H, and J) for 6 h followed by immunostaining to detect cytochrome c (Cyt. c; green), Tom20 (blue), TRAP1 (red in F), and CPOX (red in G and H). Detail images are from areas marked with white rectangles. Scale bars represent 20 µm in F–J; 2 µm in detail images in F, G, and H; and 5 µm in detail images in I and J. Arrowheads in F indicate mitochondria with high cytochrome c/low TRAP1 (red), high TRAP1/low cytochrome c (yellow), and high cytochrome c/high TRAP1 (blue). (K) Expression levels of CPOX, TRAP1, and cytochrome c in WT HeLa cells and WT, ATG5−/−, and Drp1−/− HCT116 cells was analyzed by Western blot. Tom20 was used as a loading control. Two exposures of CPOX blot are shown.

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