p62/SQSTM1 is indispensable for OXPHOS-induced mitophagy. (A) Immunoblots of cytosolic and mitochondrial fractions of HeLa cells grown in media with glucose, galactose, or acetoacetate as the sole sugar source. (B) p62 protein levels in A from three independent experiments with mean ± SD. **, P < 0.005; one-way ANOVA. (C) Live-cell imaging of HeLa cells stably expressing mCherry-p62 and stained with MitoTracker. Cells were grown in media with either glucose or acetoacetate. Scale bars, 10 µm (main), 2 µm (inset). (D) Immunoblots of cytosolic and mitochondrial fractions from WT and SAMM50 KD cells grown in glucose or galactose media. (E) Quantification of p62 recruitment to mitochondria (Mito.) in D. Values are mean ± SD from three different experiments. **, P < 0.005; *, P < 0.01; one-way ANOVA. (F) Immunoprecipitates (IP) of endogenous p62 from HeLa cells grown in glucose or galactose media analyzed for coprecipitation of endogenous SAMM50 and NIPSNAP1 by immunoblotting. (G) Quantification of coimmunoprecipitated (IP) SAMM50 in F from three independent experiments. Values are mean ± SD. **, P < 0.005; one-way ANOVA. (H and I) Immunoblots of WT and p62 KO HeLa cells grown in glucose or galactose media untreated or treated with BafA1 for 24 h (H) with quantifications from three independent experiments (I). Values are mean ± SD. **, P < 0.005; *, P < 0.01; †, NS; one-way ANOVA. (J) Live-cell images of WT and p62 KO MEFs stably expressing COX8-EGFP-mCherry grown in glucose media or glucose-free acetoacetate-containing media for 96 h. Scale bars, 20 µm. (K) Percentage of cells with red-only dots signifying mitophagy quantified in J from three independent experiments. Values are mean ± SD. ***, P < 0.001; †, NS; one-way ANOVA.