Atg8ylation participates in mTOR inactivation in response to lysosomal damage. (A) Quantification by HCM of overlaps between mTOR and LAMP2 in HeLa (WT), GBRP^TKO, and GBRP^TKO transfected with GFP-GABARAP/GABARAPL1/GABARAPL2. Cells treated with 4 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries. Yellow masks, computer-identified overlap of mTOR and LAMP2. (B) Quantification by HCM of G3BP1 puncta in HeLa (WT), GBRP^TKO, and GBRP^TKO transfected with GFP-GABARAP/GABARAPL1/GABARAPL2. Cells were treated with 4 mM LLOMe for 30 min. Red masks, computer-identified G3BP1 puncta. (C) Immunoblot analysis of GABARAP (GABA) lipidation in U2OS cells treated with 2 mM LLOMe for indicated time points. (D) Immunofluorescence confocal microscopy imaging of GFP-GABARAP and LAMP2. U2OS cells overexpressing GFP-GABARAP were treated with 2 mM LLOMe for 30 min and stained for endogenous LAMP2. Scale bar, 5 µm. (E) WB analysis of ATG9^KO, ATG3^KO in Huh7 cells. (F) WB analysis of FIP200^KO, ATG16L1^KO in Huh7 cells. (G) WB analysis of ATG3^KO, ATG13^KO in HeLa cells. (H) HCM images of Fig. 9 A. Yellow masks, computer-identified overlap of mTOR and LAMP2. (I) HCM images of Fig. 9 C. Yellow masks, computer-identified overlap of mTOR and LAMP2. (J) HCM images of Fig. 9 E. Yellow masks, computer-identified overlap of mTOR and LAMP2. (K) WB analysis of indicated proteins in ATG3^KO, ATG16L1^KO Huh7 cells. (L) Confocal microscopy imaging (i) and quantification by HCM (ii) of overlaps between mTOR and LAMP2 in parental Huh7 (WT) and ATG3 knockout Huh7 cells (ATG3^KO) transfected with scrambled siRNA as control (SCR) or NUFIP2 siRNA (NUFIP2^KD). Cells were treated with 2 mM LLOMe for 30 min. WB analysis of indicated protein in iii. (M) WB analysis of the expression of GFP-SARS-CoV-2^ORF3a in HeLa Flp-In^TetON GFP-SARS-CoV-2^ORF3a cells induced by tetracycline (Tet) for 16 h. (N) Immunoblot analysis of interaction between GCN1 and GFP-ORF3a in HEK293T Flp-In^TetON GFP-SARS-CoV-2^ORF3a cells induced by 1 µg/ml tetracycline (Tet) for 16 h. Cell lysates were immunoprecipitated with anti-GFP antibody and immunoblotted for endogenous GCN1. (O) Immunoblot analysis of interaction between GCN1 and ORF3a in HEK293T cells transfected with GFP or GFP-ORF3a. Cell lysates were immunoprecipitated with anti-GFP antibody and immunoblotted for endogenous GCN1. 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 Figs. 9 and 10. Source data are available for this figure: SourceData FS5.