Autolysosome homeostasis requires the MTMR8 PH-GRAM domain and catalytic cysteine residue. (A) Representative Western blot from HeLa cells with the indicated genotypes to detect and quantify p62 relative to actin (A′) and lipidated LC3B-II relative to cytosolic LC3B-I (A″). Asterisks indicate cell lines used in images in B, C, and F and Fig. 8. (B, C, and F) Representative maximum intensity projection of z-stack image from HeLa cells with the indicated genotypes stained with Hoechst to detect nuclei (blue), LysoTracker to detect acidic vesicles (red, depicted in B′, C′, and F′, and merged in B″, C″, and F″), and antibody to detect LAMP1 (green, depicted in B, C, and F, and merged in B″, C″, and F″). Acidified lysosomal compartments contain both LAMP1 and LysoTracker. (D and E) Quantification of the number and size of cellular LysoTracker puncta as represented in B, C, and F. Please find LAMP1 quantifications in Fig. 8, D and F. (G) Quantification of LAMP1 and LysoTracker puncta colocalization as represented in B, C, and F. (H) Representative maximum intensity projection of z-stack image from HeLa cells transfected with myc-DDK-MTMR8 and antibody stained with antibodies to detect LC3B (green, depicted in H as LC3B only, and merged in H″) and MTMR8 (magenta, white dashed line, depicted as MTMR8 only in H′, and merged in H″). (I) Representative Western blot from HeLa cells transfected with myc-DDK-MTMR8 to detect MTMR8, DDK (FLAG), p62, and LC3B. Relative levels of protein are quantified as the ratio of MTMR8 to actin (I′), FLAG to actin (I″), p62 to actin (I′″), and LC3B-II to LC3B-I (I″″). Data are presented as mean in ± SD (n = 3; A and I) and mean ± minimum to maximum (n ≥ 5 cells per 10 random images from three experimental replicates; D–G). Asterisks denote statistical significance (*, P < 0.05; **, P < 0.01; ****, P < 0.0001), using one-way ANOVA. Scale bars, 20 µm. n.s., not significant.