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. 2014 Sep 12;33(21):2473–2491. doi: 10.15252/embj.201489729

Figure 1. USP8 siRNA delays parkin recruitment onto mitochondria.

Figure 1

A, B USP8 siRNA impedes parkin recruitment onto mitochondria following CCCP treatment. U2OS-GFP-parkin cells were transfected with non-targeting or USP8 siRNA (10 nM) for 60 h (A). Untreated cells or cells treated with CCCP for 1 h were fixed, and images were acquired after staining for the mitochondrial protein TOM20. After 1-h CCCP treatment, cells were analyzed for GFP-parkin co-localization onto TOM20-positive mitochondria (B). Experiments were blinded and performed in triplicate with 100 cells analyzed for each condition. The vertical bars represent SEM for three independent experiments. For statistical analysis, a two-way ANOVA with Tukey post-test was performed, **P < 0.01.

C Validation of USP8 siRNA knockdown. U2OS-GFP-parkin cells were transfected with non-targeting or USP8 siRNA oligos (10 nM) for 60 h. Cells were lysed and analyzed by immunoblotting for USP8, parkin (long and short exposure), and actin.

D, E A delay in parkin recruitment onto mitochondria is observed in cells transfected with USP8 siRNA by live-cell microscopy. U2OS-GFP-parkin cells were transfected with non-targeting or USP8 siRNA (10 nM) for 60 h (D). 16 h prior to imaging, cells were infected with CellLight® mitochondria-RFP (Mito-RFP) to visualize mitochondria. Live-cell imaging was initiated 5 min after CCCP treatment, and images were acquired every 5 min over a 140-min period. Parkin recruitment upon membrane depolarization is visualized by the appearance of punctate GFP fluorescence superposed onto mitochondrial RFP fluorescence. Quantification of GFP-parkin recruitment to mitochondria (E) is facilitated by calculating the percentage of cells showing recruitment of GFP-parkin onto mitochondria at 5-min intervals over a period of 145 min. Experiments were performed in triplicate with 350 cells analyzed in each condition. The vertical bars represent the mean ± SEM for three independent experiments (see also Supplementary Video S1).

F, G Expression of FLAG-HA-USP8 rescues the effects of USP8 siRNA. U2OS-GFP-parkin cells were co-transfected with USP8 RNAi (5 nM) and FLAG-USP8 (0.5 μg) for 60 h (F). Cells treated with CCCP for 1 h were fixed, and images were acquired after staining for the mitochondrial protein, TOM20. After 1-h CCCP treatment, cells were analyzed for GFP-parkin co-localization onto TOM20-positive mitochondria in cells either negative or positive for FLAG-USP8 (G). Experiments were blinded and performed in triplicate with 100 cells analyzed for each condition. For statistical analysis, a two-way ANOVA with Tukey post-test was performed, ****P < 0.0001. The vertical bars represent the mean ± SEM for three independent experiments (see also Supplementary Fig S3D and E).

Source data are available online for this figure.