Figure 2.
Mosaic cytochrome c distribution is induced by abnormal accumulation of the proteins on the IMM.(A) Quantification of cells with mosaic cytochrome c in WT HCT116 cells treated with the indicated compounds for 6 h. Data are represented as mean ± SD; n = 4, n = 100 cells per condition were counted in each experiment. **, P < 0.01 versus control (all other groups were nonsignificant); one-way ANOVA with Bonferroni correction (α = 0.05). (B) Total cell lysates of WT HCT116 cells treated as indicated in the figure for 6 h were analyzed by Western blot. Tom20 was used as a loading control. (C and D) mtDNA-depleted HCT116 cells were treated with DMSO (control; C) or Acn (D) for 6 h, followed by immunostaining for cytochrome c (cyt. c; green), SDHA (red), and Tom20 (blue) and Airyscan image acquisition. Detail images are from areas outlined with white rectangles. Scale bars represent 20 µm (2 µm in detail images). (E) Number of cells with mosaic cytochrome c distribution in WT and mtDNA-depleted HCT116 cells treated with DMSO (control) or Acn for 6 h. Data represent mean ± SD; n = 4, n = 150 cells per condition per each independent experiment. (F) Total cell lysates of WT and mtDNA-depleted HCT116 cells treated with DMSO (vehicle) or Acn for 6 h were subjected to Western blotting as shown in the figure. Tom20 was used as a loading control. (G and H) WT (G) and Drp1−/− (H) HCT116 cells transfected with NDUFA12-HA were immunostained to detect cytochrome c (cyt. c; green), Tom20 (blue), and HA-tag (red). Red arrowheads in G indicate high–cytochrome c mitochondria. Detail images are from areas outlined with white rectangles. Scale bars represent 20 µm (2 µm in detail images). (I) Quantification of mosaic cytochrome c mitochondria in mock-transfected cells and cells transfected with NDUFA12-HA (A12). Data are represented as mean ± SD; n = 4, n = 150 cells per condition (WT and Drp1−/− HCT116 cells) and n = 3, n = 150 cells per condition (HeLa cells). **, P < 0.01 versus “mock” in each cell type; ns indicates nonsignificant; one-way ANOVA with Bonferroni correction (α = 0.05). (J) Expression levels of NDUFA12-HA in WT and Drp1−/− HCT116 and WT HeLa cells. Tom20 was used as a loading control. (K–P) Expression levels of Drp1 (K and N), p62 (L and O), and LC3-I/LC3II ratio (M and P) in WT HCT116 cells treated with Acn, CP, or Acn plus CP (K–M) or mtDNA-depleted HCT116 cells treated with Acn (N–P). Data are represented as mean ± SD; n = 3. *, P < 0.05 versus control; ns indicates nonsignificant; Kruskal–Wallis with Dunn post-hoc analysis (α = 0.05; K–M) or Wilcoxon rank-sum test (N–P).

Mosaic cytochrome c distribution is induced by abnormal accumulation of the proteins on the IMM. (A) Quantification of cells with mosaic cytochrome c in WT HCT116 cells treated with the indicated compounds for 6 h. Data are represented as mean ± SD; n = 4, n = 100 cells per condition were counted in each experiment. **, P < 0.01 versus control (all other groups were nonsignificant); one-way ANOVA with Bonferroni correction (α = 0.05). (B) Total cell lysates of WT HCT116 cells treated as indicated in the figure for 6 h were analyzed by Western blot. Tom20 was used as a loading control. (C and D) mtDNA-depleted HCT116 cells were treated with DMSO (control; C) or Acn (D) for 6 h, followed by immunostaining for cytochrome c (cyt. c; green), SDHA (red), and Tom20 (blue) and Airyscan image acquisition. Detail images are from areas outlined with white rectangles. Scale bars represent 20 µm (2 µm in detail images). (E) Number of cells with mosaic cytochrome c distribution in WT and mtDNA-depleted HCT116 cells treated with DMSO (control) or Acn for 6 h. Data represent mean ± SD; n = 4, n = 150 cells per condition per each independent experiment. (F) Total cell lysates of WT and mtDNA-depleted HCT116 cells treated with DMSO (vehicle) or Acn for 6 h were subjected to Western blotting as shown in the figure. Tom20 was used as a loading control. (G and H) WT (G) and Drp1−/− (H) HCT116 cells transfected with NDUFA12-HA were immunostained to detect cytochrome c (cyt. c; green), Tom20 (blue), and HA-tag (red). Red arrowheads in G indicate high–cytochrome c mitochondria. Detail images are from areas outlined with white rectangles. Scale bars represent 20 µm (2 µm in detail images). (I) Quantification of mosaic cytochrome c mitochondria in mock-transfected cells and cells transfected with NDUFA12-HA (A12). Data are represented as mean ± SD; n = 4, n = 150 cells per condition (WT and Drp1−/− HCT116 cells) and n = 3, n = 150 cells per condition (HeLa cells). **, P < 0.01 versus “mock” in each cell type; ns indicates nonsignificant; one-way ANOVA with Bonferroni correction (α = 0.05). (J) Expression levels of NDUFA12-HA in WT and Drp1−/− HCT116 and WT HeLa cells. Tom20 was used as a loading control. (K–P) Expression levels of Drp1 (K and N), p62 (L and O), and LC3-I/LC3II ratio (M and P) in WT HCT116 cells treated with Acn, CP, or Acn plus CP (K–M) or mtDNA-depleted HCT116 cells treated with Acn (N–P). Data are represented as mean ± SD; n = 3. *, P < 0.05 versus control; ns indicates nonsignificant; Kruskal–Wallis with Dunn post-hoc analysis (α = 0.05; K–M) or Wilcoxon rank-sum test (N–P).

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