Figure 1.

USP10-KD enhances dopamine-induced apoptosis.A, SH-SY5Y cells were transfected with USP10-siRNA (USP10-1, 2, or 3) or control (NT) using Lipofectamine RNAiMAX. Whole cell lysates prepared from transfected cells were characterized by Western blotting with anti-USP10 and anti-β-actin antibodies. B, SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-1 or 2) or control (siNT) using Lipofectamine RNAiMAX. The cells were then treated with 0.1 to 0.4 mM dopamine or DMSO for 12 h. The cells were treated with CCK-8 solution for 1 h. Culture supernatant was prepared from the transfected cells, and the absorbance (485 nm) of the culture supernatant was measured with an absorption meter (TriStar LB 941). The ratio of absorbance of cells to that of control siRNA (NT) treated with DMSO was presented as the mean and standard deviation (SD) of three samples. The significance of the differences was assessed by a one-way ANOVA, followed by Tukey's multiple comparison test. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. C and D, USP10-KD (siUSP10-1 or 2) and control (siNT) cells were treated with 0.4 mM dopamine or DMSO for 4 and 8 h, and cells were then stained with anticleavage caspase-3 (red) and Hoechst 33258 (blue). The staining was evaluated with a fluorescence microscope. The ratio of cells with cleaved caspase-3 relative to total cells (measured by number of nuclei) was presented as the mean and SD of five samples in (D). The significance of the difference in Figure 1D was assessed by Brown–Forsythe and Welch ANOVA followed by Dunnett's T3 multiple comparisons test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Scale bar is 10 μm. ANOVA, analysis of variance; NS, Not significant; USP10, ubiquitin-specific protease 10.