NUFIP2 and Gal8 cooperate in mTOR response to lysosomal damage. (A) Quantification by HCM of overlaps between mTOR and LAMP2 in Gal8WT^HeLa (WT) or Gal8KO^HeLa (Gal8^KO) cells treated with 2 mM LLOMe for 30 min. Yellow masks, computer-identified overlap of mTOR and LAMP2. (B) Quantification by HCM of G3BP1 puncta in Gal8WT^HeLa (WT) or Gal8KO^HeLa (Gal8^KO) cells treated with 2 mM LLOMe for 30 min. Red masks, computer-identified G3BP1 puncta. (C) HEK293T cells stably expressing FLAG-LAMTOR2 with overexpression of GFP or GFP-NUFIP2 were transfected with scrambled siRNA as control (SCR) or Gal8 siRNA (Gal8^KD). Cells were treated with 200 µM GPN for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. (D) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged NUFIP2 or G3BP1 with GST or GST-tagged Ragulator or Gal8. (E) GST pull-down assay of in vitro translated and radiolabeled Myc-tagged NUFIP2 with GST or GST-tagged Gal8. Quantification, n = 3. (F) Analysis of indicated proteins associated with lysosomes purified by anti-HA immunoprecipitation (LysoIP; TMEM192-3xHA) from HeLa WT, Gal8^KO, GABARAPs knockout (GBRP^TKO) or G3BP1 knockdown (G3BP1^KD) cells. Cells were treated with 200 µM GPN for 30 min. AR, autoradiography. 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 F6.