Figure 1. Drp1-dependent mosaic... | Rockefeller University Press

Figure 1.

$Drp1-dependent mosaic distribution of cytochrome c in cells with reduced MTF.(A, B, E, and F) WT (A and B) and Drp1−/− (E and F) HCT116 cells were treated with DMSO (control; A and E) or Acn (B and F) for 6 h, followed by immunostaining to detect cytochrome c (IMS marker, green), ClpP (matrix marker; red), and Tom20 (OMM marker; blue) and Airyscan superresolution image acquisition. Yellow arrowheads in B point to the high–cytochrome c mitochondria, and red arrowheads in B indicate the “low”–cytochrome c mitochondria. Scale bars represent 20 µm (2 µm in detail images). (C) Number of cells with high–cytochrome c mitochondria was quantified in cells treated as shown in the figure. Data represent mean ± SD of n = 3. In each experiment, triplicates of 150 cells were counted. **, P < 0.01 versus control; ns indicates nonsignificant; Kruskal–Wallis with Dunn post-hoc analysis (α = 0.05). (D) Relative area of high–cytochrome c mitochondria per cell was quantified in WT HCT116 cells treated with Acn as indicated in the figure and expressed as a percentage of total mitochondria/cell. Tom20 signal was considered as a 100%. n = 50. **, P < 0.01 versus 2 h; ^, P < 0.01 versus 4 h; one-way ANOVA with Bonferroni correction (α = 0.05). (G) Quantification of cells with mosaic cytochrome c distribution. Cells indicated in the figure were treated with DMSO (vesicle; control) or Acn for 6 h. Data represent mean ± SD; n = 3, n = 100 cells per condition per each N. **, P < 0.01 versus WT Acn in each cell type; ns indicates nonsignificant; one-way ANOVA with Bonferroni correction (α = 0.05). (H) Overlap between cytochrome c and ClpP in control and Acn-treated WT, Drp1−/−, and Bax/Bak DKO HCT116 cells was estimated from maximum intensity projection images (as in A, B, E, and F). The values are shown as Pearson’s correlation coefficient. Data are represented as mean ± SD; n = 30 cells per condition (data pulled from n = 2 independent experiments). **, P < 0.01 versus control in each group; ns indicates nonsignificant; one-way ANOVA with Bonferroni correction (α = 0.05). (I and J) WT (I) and Mff−/− (J) HCT116 cells treated as indicated in the figure were subjected to cell fractionation and Western blot analysis to detect subcellular localization of Drp1. Tom40 was used as a loading control. C, cytosol (postmitochondrial supernatants, including LMs); Cyt. C, cytochrome c; M, mitochondria-enriched HM fractions, T, total cell lysate.$

Drp1-dependent mosaic distribution of cytochrome c in cells with reduced MTF. (A, B, E, and F) WT (A and B) and Drp1^−/− (E and F) HCT116 cells were treated with DMSO (control; A and E) or Acn (B and F) for 6 h, followed by immunostaining to detect cytochrome c (IMS marker, green), ClpP (matrix marker; red), and Tom20 (OMM marker; blue) and Airyscan superresolution image acquisition. Yellow arrowheads in B point to the high–cytochrome c mitochondria, and red arrowheads in B indicate the “low”–cytochrome c mitochondria. Scale bars represent 20 µm (2 µm in detail images). (C) Number of cells with high–cytochrome c mitochondria was quantified in cells treated as shown in the figure. Data represent mean ± SD of n = 3. In each experiment, triplicates of 150 cells were counted. **, P < 0.01 versus control; ns indicates nonsignificant; Kruskal–Wallis with Dunn post-hoc analysis (α = 0.05). (D) Relative area of high–cytochrome c mitochondria per cell was quantified in WT HCT116 cells treated with Acn as indicated in the figure and expressed as a percentage of total mitochondria/cell. Tom20 signal was considered as a 100%. n = 50. **, P < 0.01 versus 2 h; ^, P < 0.01 versus 4 h; one-way ANOVA with Bonferroni correction (α = 0.05). (G) Quantification of cells with mosaic cytochrome c distribution. Cells indicated in the figure were treated with DMSO (vesicle; control) or Acn for 6 h. Data represent mean ± SD; n = 3, n = 100 cells per condition per each N. **, P < 0.01 versus WT Acn in each cell type; ns indicates nonsignificant; one-way ANOVA with Bonferroni correction (α = 0.05). (H) Overlap between cytochrome c and ClpP in control and Acn-treated WT, Drp1^−/−, and Bax/Bak DKO HCT116 cells was estimated from maximum intensity projection images (as in A, B, E, and F). The values are shown as Pearson’s correlation coefficient. Data are represented as mean ± SD; n = 30 cells per condition (data pulled from n = 2 independent experiments). **, P < 0.01 versus control in each group; ns indicates nonsignificant; one-way ANOVA with Bonferroni correction (α = 0.05). (I and J) WT (I) and Mff^−/− (J) HCT116 cells treated as indicated in the figure were subjected to cell fractionation and Western blot analysis to detect subcellular localization of Drp1. Tom40 was used as a loading control. C, cytosol (postmitochondrial supernatants, including LMs); Cyt. C, cytochrome c; M, mitochondria-enriched HM fractions, T, total cell lysate.