Figure 3.
Time-lapse imaging of cyt. c-GFP in Acn-treated WT and Drp1−/− cells.(A–C) WT (A and C) and Drp1−/− (B) HeLa cells cotransfected with cyt. c-GFP (green in A–C) and mito-RFP (red in A–C) treated with Acn (A and B) or FCCP (C) were subjected to time-lapse imaging for up to 300 min. Detail images at time 0 min and 300 min in A–C are from areas outlined with green rectangles. Arrowheads in A point to high cyt. c-GFP intensity mitochondria and corresponding mito-RFP signal. Scale bars represent 20 µm (2 µm in detail images) in A and B and 10 µm (1 µm in detail images) in C. (D–F) Formation of high cyt. c-GFP intensity mitochondria in WT (D), Drp1−/− (E), and Mff−/− (F) HeLa cells over time in several time-lapse experiments were quantified. Data represent n = 3 (6 cells) for control WT cells, n = 8 (11 cells) for Acn-treated WT cells, n = 5 (8 cells) for Acn plus CP-treated WT cells, n = 5 (8 cells) for FCCP-treated cells, n = 4 (10 cells) for Acn-treated Parkin-mCherry–expressing WT cells, n = 6 (11 cells) for Acn-treated Drp1−/− cells, n = 5 (10 cells) for Drp1-mCherry–expressing Acn-treated Drp1−/− cells, and n = 5 (8 cells) for Acn-treated Mff−/− cells. **, P < 0.01 versus control at 300 min; ns indicates nonsignificant; one-way ANOVA with Bonferroni correction (α = 0.05). To facilitate data evaluation in D, the values for control were shifted 0.5 units down, and the values for Acn plus CP were shifted 0.5 units up. (G) Fluorescence intensity of TMRM in WT cells treated with Acn or Acn plus CP for up to 300 min was quantified and plotted as function of time. The TMRM fluorescence values at time 0 min (the first image in each series) were taken as 1. Data represent n = 3 (n = 10). **, P < 0.01 at 300 min; two-tailed Student’s t test. (H) Patterns of cyt. c-GFP (green) and TMRM (red) localization and intensity in Acn-treated WT cells. The same area of a typical cell at 0 min and at 280 min with Acn is shown. Arrowheads point to some high cyt. c-GFP intensity mitochondria and corresponding TMRM signal. Scale bars represent 3 µm.

Time-lapse imaging of cyt. c-GFP in Acn-treated WT and Drp1−/− cells. (A–C) WT (A and C) and Drp1−/− (B) HeLa cells cotransfected with cyt. c-GFP (green in A–C) and mito-RFP (red in A–C) treated with Acn (A and B) or FCCP (C) were subjected to time-lapse imaging for up to 300 min. Detail images at time 0 min and 300 min in A–C are from areas outlined with green rectangles. Arrowheads in A point to high cyt. c-GFP intensity mitochondria and corresponding mito-RFP signal. Scale bars represent 20 µm (2 µm in detail images) in A and B and 10 µm (1 µm in detail images) in C. (D–F) Formation of high cyt. c-GFP intensity mitochondria in WT (D), Drp1−/− (E), and Mff−/− (F) HeLa cells over time in several time-lapse experiments were quantified. Data represent n = 3 (6 cells) for control WT cells, n = 8 (11 cells) for Acn-treated WT cells, n = 5 (8 cells) for Acn plus CP-treated WT cells, n = 5 (8 cells) for FCCP-treated cells, n = 4 (10 cells) for Acn-treated Parkin-mCherry–expressing WT cells, n = 6 (11 cells) for Acn-treated Drp1−/− cells, n = 5 (10 cells) for Drp1-mCherry–expressing Acn-treated Drp1−/− cells, and n = 5 (8 cells) for Acn-treated Mff−/− cells. **, P < 0.01 versus control at 300 min; ns indicates nonsignificant; one-way ANOVA with Bonferroni correction (α = 0.05). To facilitate data evaluation in D, the values for control were shifted 0.5 units down, and the values for Acn plus CP were shifted 0.5 units up. (G) Fluorescence intensity of TMRM in WT cells treated with Acn or Acn plus CP for up to 300 min was quantified and plotted as function of time. The TMRM fluorescence values at time 0 min (the first image in each series) were taken as 1. Data represent n = 3 (n = 10). **, P < 0.01 at 300 min; two-tailed Student’s t test. (H) Patterns of cyt. c-GFP (green) and TMRM (red) localization and intensity in Acn-treated WT cells. The same area of a typical cell at 0 min and at 280 min with Acn is shown. Arrowheads point to some high cyt. c-GFP intensity mitochondria and corresponding TMRM signal. Scale bars represent 3 µm.

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