Figure 1.
Lysosomal damage induces SG formation. (A) DIA LC/MS/MS quantitative analysis of proteins associated with lysosomes purified by LysoIP (anti-HA IP) from HEK293T cells expressing TMEM192-3xHA untreated or treated with 1 mM LLOMe for 30 min. Three groups of proteins are denoted: ESCRT components, green; autophagy factors, blue; SG components, purple. Scatter (volcano) plot shows log2 fold changes and −Log10 P values; n = 3 (see Table S1, Tab 1). Dashed line, significance cut-off (P < 0.05). (B) Protease accessibility analysis of proteins associated with purified lysosomes (LysoIP). Huh7 cells were treated with 2 mM LLOMe. LysoIP preparations (treated or not with detergent Triton X-100) were digested with 30 µg/ml proteinase K for 30 min and analyzed by immunoblotting. (C) Quantification by HCM of G3BP1 puncta. U2OS cells were treated with EBSS, 4 mM LOMe, 2 mM LLOMe, 200 µM GPN, or 400 µg/ml silica for 30 min. White masks, algorithm-defined cell boundaries (primary objects); red masks, computer-identified G3BP1 puncta (target objects). (D) Fluorescence confocal microscopy imaging of G3BP1. U2OS cells were treated with 2 mM LLOMe for 30 min and immunostained for endogenous G3BP1. Scale bar, 5 µm. (E) Quantification by HCM of G3BP1 puncta in BMM cells treated with 2 mM LLOMe or 100 µM NaAsO2 for 2 h. Green masks, computer-identified G3BP1 puncta. (F) Quantification by HCM of G3BP1 puncta in U2OS cells treated with LLOMe at indicated doses or 100 µM NaAsO2 in the presence or absence of 10 µg/ml cycloheximide (CHX) for 30 min. HCM images in Fig. S1 G. (G) Analysis of proteins associated with purified lysosomes (LysoIP; TMEM192-3xHA) from HEK293T cells treated with 2 mM LLOMe in the presence or absence of 10 µg/ml CHX for 30 min. TMEM192-2xFLAG, control. (H) Immunoblot analysis of eIF2α (S51) phosphorylation in BMM cells treated with 2 mM LLOMe or 100 µM NaAsO2 for 2 h; eIF2α p-S51 quantification, n = 3. (I) Confocal microscopy analysis of G3BP1 (Alexa Fluor 488) and polyA RNA (Cy3-oligo[dT]) by FISH in U2OS cells treated with 2 mM LLOMe for 30 min. Scale bar, 5 µm. (J) HCM analysis of protein synthesis using Click-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay (Thermo Fisher Scientific) in U2OS cells treated with LLOMe at indicated doses or 100 µM NaAsO2 or 10 µg/ml CHX for 30 min. (K) Immunoblot analysis of ATF4 and phosphorylation of 4EBP1 (S65) and eIF2α (S51) in U2OS cells treated with 2 mM LLOMe for indicated time points; quantification of ATF4 and phosphorylation of 4EBP1 (S65) and eIF2α (S51), n = 3. 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 < 0.05; **, P < 0.01, ANOVA. See also Fig. S1. Source data are available for this figure: SourceData F1.

Lysosomal damage induces SG formation. (A) DIA LC/MS/MS quantitative analysis of proteins associated with lysosomes purified by LysoIP (anti-HA IP) from HEK293T cells expressing TMEM192-3xHA untreated or treated with 1 mM LLOMe for 30 min. Three groups of proteins are denoted: ESCRT components, green; autophagy factors, blue; SG components, purple. Scatter (volcano) plot shows log2 fold changes and −Log10 P values; n = 3 (see Table S1, Tab 1). Dashed line, significance cut-off (P < 0.05). (B) Protease accessibility analysis of proteins associated with purified lysosomes (LysoIP). Huh7 cells were treated with 2 mM LLOMe. LysoIP preparations (treated or not with detergent Triton X-100) were digested with 30 µg/ml proteinase K for 30 min and analyzed by immunoblotting. (C) Quantification by HCM of G3BP1 puncta. U2OS cells were treated with EBSS, 4 mM LOMe, 2 mM LLOMe, 200 µM GPN, or 400 µg/ml silica for 30 min. White masks, algorithm-defined cell boundaries (primary objects); red masks, computer-identified G3BP1 puncta (target objects). (D) Fluorescence confocal microscopy imaging of G3BP1. U2OS cells were treated with 2 mM LLOMe for 30 min and immunostained for endogenous G3BP1. Scale bar, 5 µm. (E) Quantification by HCM of G3BP1 puncta in BMM cells treated with 2 mM LLOMe or 100 µM NaAsO2 for 2 h. Green masks, computer-identified G3BP1 puncta. (F) Quantification by HCM of G3BP1 puncta in U2OS cells treated with LLOMe at indicated doses or 100 µM NaAsO2 in the presence or absence of 10 µg/ml cycloheximide (CHX) for 30 min. HCM images in Fig. S1 G. (G) Analysis of proteins associated with purified lysosomes (LysoIP; TMEM192-3xHA) from HEK293T cells treated with 2 mM LLOMe in the presence or absence of 10 µg/ml CHX for 30 min. TMEM192-2xFLAG, control. (H) Immunoblot analysis of eIF2α (S51) phosphorylation in BMM cells treated with 2 mM LLOMe or 100 µM NaAsO2 for 2 h; eIF2α p-S51 quantification, n = 3. (I) Confocal microscopy analysis of G3BP1 (Alexa Fluor 488) and polyA RNA (Cy3-oligo[dT]) by FISH in U2OS cells treated with 2 mM LLOMe for 30 min. Scale bar, 5 µm. (J) HCM analysis of protein synthesis using Click-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay (Thermo Fisher Scientific) in U2OS cells treated with LLOMe at indicated doses or 100 µM NaAsO2 or 10 µg/ml CHX for 30 min. (K) Immunoblot analysis of ATF4 and phosphorylation of 4EBP1 (S65) and eIF2α (S51) in U2OS cells treated with 2 mM LLOMe for indicated time points; quantification of ATF4 and phosphorylation of 4EBP1 (S65) and eIF2α (S51), n = 3. 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 < 0.05; **, P < 0.01, ANOVA. See also Fig. S1. Source data are available for this figure: SourceData F1.

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