Figure 8.
Effects of reduced MTF in Parkin-overexpressing WT and Drp1−/− cells.(A) WT HeLa cells cotransfected with cyt. c-GFP (green) and Parkin-Cherry (red) were treated with Acn and subjected to time-lapse live-cell imaging. Arrowheads indicate high–cyt. c-GFP mitochondria and the corresponding Parkin-Cherry. Scale bar represents 10 µm. (B and C) WT (B) and Drp1−/− (C) HCT116 cells transfected with Parkin-YFP (green in B and C), followed by treatment with Acn for 6 h, and immunostaining to detect conjugated Ub (FK2; blue) and Tom20 (red). Detail images in B and C are from areas outlined with rectangles. Scale bars represent 20 µm (5 µm in detail images). (D) Parkin-YFP–expressing WT and Drp1−/− cells treated with DMSO (control), Acn, CP, and Acn plus CP for 6 h were quantified for mitochondrial localization of Parkin. Parkin-YFP–expressing cells were counted. Data represent mean ± SD; n = 5 (n = 100). (E and F) WT (E) and ATG5−/− (F) HCT116 cells transfected with Parkin-YFP (green) were treated with Acn for 6 h and immunostained to detect cytochrome c (blue) and Tom20 (red). Parkin-YFP–expressing cells are traced with yellow lines. Scale bars represent 20 µm. (G) Number of high–cytochrome c mitochondria per cell was blindly quantified in Parkin-YFP (P+)–expressing (P+) and deficient (P−) WT and ATG5−/− cells treated with Acn. P+ and P− cells were from the same samples. n = 50 cells/condition. **, P < 0.01versus WT P−; ns indicates nonsignificant; one-way ANOVA with Bonferroni correction (α = 0.05). (H) Submitochondrial localization of Parkin-YFP (green) and Ub (blue) in Acn-treated cells. Scale bars represent 5 µm. (I) Quantification of relative position of OMM (Tom20), IMM (Tim23), and Parkin-YFP versus the Ub (FK2) signal. n = 60 per condition. Data pulled from n = 3 independent experiments. To facilitate data evaluation, the values were slightly right-shifted for Parkin-YFP and slightly left-shifted for Tom20. (J and K) Untransfected (J) and Parkin-YFP transfected (K) WT HCT116 cells were treated with Acn for 6 h, followed by immunostaining to detect Ub (FK2; blue in merged images) and phosphorylated Ub (phosphor-Ub; green in merged images). YFP-Parkin is pseudocolored in red (K). Detail images in J and K are from areas marked with rectangles from cells overlaid with yellow lines. Scale bars represent 20 µm (2 µm in detail images).

Effects of reduced MTF in Parkin-overexpressing WT and Drp1−/− cells. (A) WT HeLa cells cotransfected with cyt. c-GFP (green) and Parkin-Cherry (red) were treated with Acn and subjected to time-lapse live-cell imaging. Arrowheads indicate high–cyt. c-GFP mitochondria and the corresponding Parkin-Cherry. Scale bar represents 10 µm. (B and C) WT (B) and Drp1−/− (C) HCT116 cells transfected with Parkin-YFP (green in B and C), followed by treatment with Acn for 6 h, and immunostaining to detect conjugated Ub (FK2; blue) and Tom20 (red). Detail images in B and C are from areas outlined with rectangles. Scale bars represent 20 µm (5 µm in detail images). (D) Parkin-YFP–expressing WT and Drp1−/− cells treated with DMSO (control), Acn, CP, and Acn plus CP for 6 h were quantified for mitochondrial localization of Parkin. Parkin-YFP–expressing cells were counted. Data represent mean ± SD; n = 5 (n = 100). (E and F) WT (E) and ATG5−/− (F) HCT116 cells transfected with Parkin-YFP (green) were treated with Acn for 6 h and immunostained to detect cytochrome c (blue) and Tom20 (red). Parkin-YFP–expressing cells are traced with yellow lines. Scale bars represent 20 µm. (G) Number of high–cytochrome c mitochondria per cell was blindly quantified in Parkin-YFP (P+)–expressing (P+) and deficient (P−) WT and ATG5−/− cells treated with Acn. P+ and P− cells were from the same samples. n = 50 cells/condition. **, P < 0.01versus WT P−; ns indicates nonsignificant; one-way ANOVA with Bonferroni correction (α = 0.05). (H) Submitochondrial localization of Parkin-YFP (green) and Ub (blue) in Acn-treated cells. Scale bars represent 5 µm. (I) Quantification of relative position of OMM (Tom20), IMM (Tim23), and Parkin-YFP versus the Ub (FK2) signal. n = 60 per condition. Data pulled from n = 3 independent experiments. To facilitate data evaluation, the values were slightly right-shifted for Parkin-YFP and slightly left-shifted for Tom20. (J and K) Untransfected (J) and Parkin-YFP transfected (K) WT HCT116 cells were treated with Acn for 6 h, followed by immunostaining to detect Ub (FK2; blue in merged images) and phosphorylated Ub (phosphor-Ub; green in merged images). YFP-Parkin is pseudocolored in red (K). Detail images in J and K are from areas marked with rectangles from cells overlaid with yellow lines. Scale bars represent 20 µm (2 µm in detail images).

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