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. 2022 Sep 30;221(11):e202207091. doi: 10.1083/jcb.202207091

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© 2022 Jia et al.

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Figure S1. — SG formation during lysosomal damage. (A) Immunoblot analysis of proteins associated with purified lysosomes (LysoIP; anti-HA immunoprecipitation TMEM192^3xHA) from HEK293T cells treated with 2 mM LLOMe or 100 µM NaAsO₂ for 30 min. TMEM192^2xFLAG, control. (B) Quantification by HCM of DCP1a and G3BP1 puncta in U2OS cells treated with 2 mM LLOMe for 30 min. PB, P-body. (C) Quantification by HCM of G3BP1 puncta in Huh7 cells treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries (primary objects); Green masks, computer-identified G3BP1 puncta (target objects). (D) Quantification by HCM of Gal3 puncta in BMM cells treated with 2 mM LLOMe or 100 µM NaAsO₂ for 2 h. Red masks, computer-identified galectin-3 puncta. (E) Quantification by HCM of TIA1 puncta in U2OS cells treated with 2 mM LLOMe for 30 min. Red masks, computer-identified TIA1 puncta. (F) Quantification by HCM of TIA1 puncta in HeLa cells treated with 4 mM LLOMe for 30 min. Red masks, computer-identified TIA1 puncta. (G) Quantification by HCM of G3BP1 and Gal3 puncta in U2OS cells treated with increasing doses of LLOMe or 100 µM NaAsO₂ in the presence or absence of 10 µg/ml cycloheximide (CHX) for 30 min. (i) HCM sample images corresponding to Fig. 1 F. Red masks, computer-identified G3BP1 puncta. (ii and ⅲ) Green masks, computer-identified Gal3 puncta and corresponding quantification in iii. (H) Immunoblot analysis of eIF2α (S51) phosphorylation in U2OS cells treated with 2 mM LLOMe for 30 min and followed by 1 h washout. (I) Quantification by HCM of G3BP1 puncta in U2OS cells treated with 2 mM LLOMe for 30 min and followed by 1 h washout. Red masks, computer-identified G3BP1 puncta. (J) Immunoblot analysis of eIF2α (S51) phosphorylation in HEK293T cells treated with 2 mM LLOMe or 100 µM NaAsO₂ for 30 min. (K) Schematic summary of the findings in Fig. 1. (L) Quantification by HCM of Lysotracker Red (LTR) and G3BP1 puncta in parental HeLa WT and Gal3^KO cells treated with 4 mM LLOMe for 30 min. Red masks, computer-identified LTR puncta. Green masks, computer-identified G3BP1 puncta. (M) Quantification by HCM of poly(A) RNA (Cy3-oligo[dT]) in U2OS cells transfected with scrambled siRNA as control (SCR) or G3BP1/2 siRNA for single or double knockdown (DKD). Cells were treated with 2 mM LLOMe for 30 min. Red masks, computer-identified poly(A) RNA puncta. 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. 1. Source data are available for this figure: SourceData FS1.