Fig. 1.

CRISPR/Cas9 knockout of Gp78 in HT-1080 prevents damage-induced mitophagy. A Schematic showing Exon 1 region of Gp78 gene on chromosome 16 and location of gRNA1 targeting the start codon, deleting ATG (∆G or inserting extra T), and of gRNA2, inserting an extra T at amino acid 16 causing frameshift and termination. Western blot for Gp78 is shown for wild-type HT-1080 cells and all six gRNA1 and gRNA2 Gp78 knockout CRISPR clones with β-actin as a loading control (full blots shown in Supp. Figure 5). B Wild-type HT-1080 cells and the six Gp78 knockout CRISPR clones were incubated with either DMSO or 10 μM CCCP for 24 h then fixed and labeled for mitochondrial ATP synthase subunit β and imaged by 3D spinning disk confocal microscopy. Representative images of HT-1080 and a CRISPR clone including magnification of boxed region are shown. Quantification of total mitochondrial volume and average volume per mitochondrial segment are shown in the bar graphs (n = 3 independent biological replicates; > 10 cells/condition per experiment; *, p < 0.05; **, p < 0.01; ***, p < 0.001; for total mitochondrial volume graph (left), CCCP treated HT-1080 cells relative to DMSO treated HT-1080 cells, DMSO treated Gp78 KO clones relative to DMSO-treated HT-1080 cells and CCCP-treated Gp78 KO clones relative to CCCP-treated HT-1080 cells; Mean ± SEM; Scale Bars: 5 μm; 1 μm for zooms)