NUFIP2 contributes to mTOR inactivation during lysosomal damage. (A) Immunoblot analysis of FLAG-NUFIP2 or FLAG-NUFIP2ΔNLS associated with purified lysosomes (LysoIP; TMEM192-3xHA). Huh7 cells transfected with FLAG-NUFIP2 or FLAG-NUFIP2ΔNLS, treated or not with 2 mM LLOMe for 30 min. TMEM192-2xFLAG, control. (B) Quantification by HCM of G3BP1 puncta in parental Huh7 (WT) and NUFIP2-knockout Huh7 cells (NUFIP2^KO) treated with 2 mM LLOMe or 100 µM NaAsO₂ for 30 min. Red masks, computer-identified G3BP1 puncta. (C) Quantification by HCM of overlaps and confocal microscopy imaging of mTOR and LAMP2 in parental Huh7 (WT) and NUFIP2-knockout Huh7 cells (NUFIP2^KO) treated with 2 mM LLOMe for 30 min. HCM images in Fig. S3 H. Scale bar, 5 µm. (D) Immunoblot analysis of indicated proteins in parental Huh7 (WT) and NUFIP2-knockout Huh7 cells (NUFIP2^KO) treated with 2 mM LLOMe for 30 min. The level of phosphorylation of ULK1 (S757), S6K (T389), and 4EBP1 (S65) was quantified based on three independent experiments. (E) Immunoblot analysis of proteins associated with purified lysosomes (LysoIP) from HEK293T cells treated with 1 μM PP242 for 2 h or 2 mM LLOMe for 30 min or HEK293T cells stably expressing constitutively active RagB GTPase (RagB^Q99L) treated with 2 mM LLOMe for 30 min. 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. S3. Source data are available for this figure: SourceData F4.