Atg8ylation plays a role in mTOR inhibition and competes with SG formation during lysosomal damage. (A) Quantification by HCM and confocal microscopy imaging of overlaps between mTOR and LAMP2 in parental Huh7 (WT), ATG9^KO, and ATG3^KO treated with 2 mM LLOMe for 30 min. HCM images in Fig. S5 H. Scale bar, 5 µm. (B) Quantification by HCM of G3BP1 puncta. Parental Huh7 (WT), ATG9^KO, and ATG3^KO were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries. Red masks, computer-identified G3BP1 puncta. (C) Quantification by HCM and confocal microscopy imaging of overlaps between mTOR and LAMP2 in parental Huh7 (WT), FIP200^KO, and ATG16L1^KO treated with 2 mM LLOMe for 30 min. HCM images in Fig. S5 I. Scale bar, 5 µm. (D) Quantification by HCM of G3BP1 puncta. Parental Huh7 (WT), FIP200^KO, and ATG16L1^KO were treated with 2 mM LLOMe for 30 min. Red masks, computer-identified G3BP1 puncta. (E) Quantification by HCM and confocal microscopy imaging of overlaps between mTOR and LAMP2 in parental HeLa (WT), ATG13^KO, and ATG3^KO treated with 4 mM LLOMe for 30 min. HCM images in Fig. S5 J. Scale bar, 5 µm. (F) Quantification by HCM of G3BP1 puncta. Parental HeLa (WT), ATG13^KO, and ATG3^KO were treated with 4 mM LLOMe for 30 min. Red masks, computer-identified G3BP1 puncta. (G) Schematic summary of the findings in this study. Ctrl, control (untreated cells). Data, means ± SEM; HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). †, P ≥ 0.05 (not significant); **, P < 0.01, ANOVA. See also Fig. S5. Source data are available for this figure: SourceData F9.