Figure S3.
NUFIP2 exits nucleus and localizes to lysosomes upon damage and cooperates with Gal8 in mTORC1 response to lysosomal damage. (A) Immunofluorescence confocal microscopy analysis of G3BP1 and NUFIP2. Huh7 cells were treated with 2 mM LLOMe for 30 min and stained for endogenous G3BP1 and NUFIP2. Scale bar, 5 µm. (B) The NLS analysis of NUFIP2 by cNLS Mapper. The sequence in red, predicted NLS in NUFIP2, was deleted for generating NUFIP2ΔNLS. (C) Immunoblot analysis of NUFIP2 distribution in nuclear or postnuclear of Huh7 cells transfected with FLAG-NUFIP2 or NUFIP2ΔNLS after the treatment with 2 mM LLOMe for 30 min. (D and E) Confocal microscopy analysis (D) and quantification by HCM (E) of overlaps between mTOR and LAMP2 in U2OS transfected with scrambled siRNA as control (SCR) or NUFIP2 siRNA (NUFIP2KD) treated with 2 mM LLOMe for 30 min. Scale bar, 5 µm. (F) Immunoblot analysis of indicated proteins in U2OS cells transfected with scrambled siRNA as control (SCR) or NUFIP2 siRNA (NUFIP2KD) 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. (G) Immunoblot validation of NUFIP2-knockout in Huh7 cells. #E7 was used in the following experiments, named as Huh7NUFIP2-KO. (H) HCM images of Fig. 4C. Yellow masks, computer-identified overlap of mTOR and LAMP2. (I) Immunoblot analysis of indicated proteins in Huh7 cells transfected with scrambled siRNA as control (SCR) or TIA1 siRNA (TIA1KD) treated with 2 mM LLOMe for 30 min. (J) Analysis of indicated proteins associated with lysosomes purified by anti-HA immunoprecipitation (LysoIP; TMEM192-3xHA) from HEK293T cells treated with 2 mM LLOMe for 30 min. TMEM192-2xFLAG, control. (K) Immunoblot analysis of the phosphorylation of ULK1 (S757), S6K1 (T389) and 4EBP1 (S65) in parental HeLa (WT) and TSC2-knockout HeLa cells (TSC2KO) treated with 2 mM LLOMe for 30 min. (L) Immunoblot analysis of the phosphorylation of ULK1 (S757), S6K1 (T389), and 4EBP1 (S65) in HEK293T cells or HEK293T cells stably expressing constitutively active RagB GTPase (RagBQ99L) treated with 2 mM LLOMe for 30 min. (M) GST pull-down assay of in vitro translated and radiolabeled Myc-tagged NUFIP2 or G3BP1 with GST or GST-tagged Gal8. 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; **, P < 0.01, ANOVA. See also Figs. 4, 5, and 6. Source data are available for this figure: SourceData FS3.

NUFIP2 exits nucleus and localizes to lysosomes upon damage and cooperates with Gal8 in mTORC1 response to lysosomal damage. (A) Immunofluorescence confocal microscopy analysis of G3BP1 and NUFIP2. Huh7 cells were treated with 2 mM LLOMe for 30 min and stained for endogenous G3BP1 and NUFIP2. Scale bar, 5 µm. (B) The NLS analysis of NUFIP2 by cNLS Mapper. The sequence in red, predicted NLS in NUFIP2, was deleted for generating NUFIP2ΔNLS. (C) Immunoblot analysis of NUFIP2 distribution in nuclear or postnuclear of Huh7 cells transfected with FLAG-NUFIP2 or NUFIP2ΔNLS after the treatment with 2 mM LLOMe for 30 min. (D and E) Confocal microscopy analysis (D) and quantification by HCM (E) of overlaps between mTOR and LAMP2 in U2OS transfected with scrambled siRNA as control (SCR) or NUFIP2 siRNA (NUFIP2KD) treated with 2 mM LLOMe for 30 min. Scale bar, 5 µm. (F) Immunoblot analysis of indicated proteins in U2OS cells transfected with scrambled siRNA as control (SCR) or NUFIP2 siRNA (NUFIP2KD) 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. (G) Immunoblot validation of NUFIP2-knockout in Huh7 cells. #E7 was used in the following experiments, named as Huh7NUFIP2-KO. (H) HCM images of Fig. 4C. Yellow masks, computer-identified overlap of mTOR and LAMP2. (I) Immunoblot analysis of indicated proteins in Huh7 cells transfected with scrambled siRNA as control (SCR) or TIA1 siRNA (TIA1KD) treated with 2 mM LLOMe for 30 min. (J) Analysis of indicated proteins associated with lysosomes purified by anti-HA immunoprecipitation (LysoIP; TMEM192-3xHA) from HEK293T cells treated with 2 mM LLOMe for 30 min. TMEM192-2xFLAG, control. (K) Immunoblot analysis of the phosphorylation of ULK1 (S757), S6K1 (T389) and 4EBP1 (S65) in parental HeLa (WT) and TSC2-knockout HeLa cells (TSC2KO) treated with 2 mM LLOMe for 30 min. (L) Immunoblot analysis of the phosphorylation of ULK1 (S757), S6K1 (T389), and 4EBP1 (S65) in HEK293T cells or HEK293T cells stably expressing constitutively active RagB GTPase (RagBQ99L) treated with 2 mM LLOMe for 30 min. (M) GST pull-down assay of in vitro translated and radiolabeled Myc-tagged NUFIP2 or G3BP1 with GST or GST-tagged Gal8. 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; **, P < 0.01, ANOVA. See also Figs. 4, 5, and 6. Source data are available for this figure: SourceData FS3.

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