Fig. 3

The ubiquitin kinase PINK1 was activated in CuONPs-treated cells. A Western blotting analysis and quantification of the protein levels of PINK1 and p-ubiquitin (Ser65) in EA.hy926 cells treated with CuONPs (0, 5, 10, 15 and 20 μg/ml) for 12 h. Actin was used as internal control. B Representative fluorescence images of colocalization of PINK1-GFP with Mito-DsRed in EA.hy926 treated with CuONPs (10 μg/ml). White arrows indicate PINK1-positive dots that colocalized with Mito-DsRed signals. Scale bars, 20 μm. C RT-qPCR analysis was performed to verify the knockdown efficiency of PINK1 siRNA. D MTS analysis was performed to detect EA.hy926 cell viability. The cells were transfected with siNC or siPINK1 for 48 h and then treated with CuONPs (10 μg/ml) for 12 h. E and F Representative FCM results of EA.hy926 cells labeled with MitoTracker Green FM (E) and MitoSOX (F). MFI, mean fluorescence intensity. In A and C, two-tailed unpaired Student’s t test was performed for statistical analysis. In D, E and F, one-way ANOVA with Tukey’s test was used for multiple comparisons. Results are representative of at least three independent experiments. Data are mean ± S.D. *p < 0.05