Fig. 3

High content assay for Parkin translocation. Effect of PINK1 shRNA (a, b) and GPATCH2L shRNA (c, d). a, c Cells are labeled for nuclei (blue; Hoechst), Parkin-GFP (green), mitochondria (red, Mitotracker Deepred). Untreated cells infected with shRNA encoding a scrambled sequence show absence of puncta (left panel). Cells infected with a scrambled sequence but treated with CCCP show a significant increase in puncta formation (middle panel). Infection of cells with shRNA targeting PINK1 or GPATCH2L prevents the accumulation of Parkin on mitochondrial (right panel). b, d The graph displays the normalized ratio of cells positive for translocation and cells negative for parkin translocation. All values were normalized to the negative control (CCCP treated infected with shRNA encoding a scrambled sequence). Data are median values ± median absolute deviation (MAD) of N = 6 measurements. *p < 0.05, **p < 0.01, and ***p < 0.001, Mann–Whitney U test (see “Methods”). All shRNA clones that meet the cutoff criteria (see “Methods”) are shown