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. 2015 Mar 4;16(5):618–627. doi: 10.15252/embr.201439820

Figure 3. USP30 depletion enhances CCCP-induced Parkin-dependent cell death.

Figure 3

  1. hTERT-RPE1-YFP-Parkin cells were transfected with non-targeting (NT1) or two individual USP30-targeting siRNA oligos (D1, D3) for 72 h and treated with CCCP for 4 h. PARP-p85 (normalized to actin) is shown as a mean ± SD of four independent experiments. One-way ANOVA, Dunnett's test, ***P = 0.001.
  2. hTERT-RPE1-YFP-Parkin cells were transfected and treated as in (A), and cells were imaged every 15 min in the presence of the membrane-impermeable dye DRAQ7 (pink). Shown is a cropped single frame from a representative video. YFP-Parkin shown in grey. Scale bar: 10 μm. Graph shows averaged data from three independent experiments ± SD. One-way ANOVA, Dunnett's test, *P = 0.05.
  3. hTERT-RPE1-YFP-Parkin cells were transfected with siRNA against PINK1 and USP30 (D1) and treated with CCCP. Shown are results from a representative experiment.
  4. hTERT-RPE1-YFP-Parkin cells and hTERT-RPE1 parental cells were transfected with NT1 or USP30 siRNA D1 and treated with CCCP. Shown are results from a representative experiment.
  5. HEK293T cells expressing GFP-tagged USP30 were lysed and subjected to immunoprecipitation (IP) with anti-GFP and probed as indicated. 1.8% of input sample loaded. U30, USP30-GFP; CS, catalytically inactive mutant USP30-C77S-GFP.
  6. hTERT-RPE1-YFP-Parkin cells were transfected with NT1 or USP30 siRNA (D1, D3) and then treated for 5 h with CCCP together with either epoxomicin (100 nM) or folimycin (100 nM).
  7. Schematic diagram illustrating the role of the proteasome and USP30 in opposing mitophagy and CCCP-induced cell death in Parkin-overexpressing RPE1 cells.

Source data are available online for this figure.