Fig 2: Catalytic activity of GCN2 is important for SG formation. (A–C) U2OS cells were transfected with siRNA-2 targeting GCN2 (2), non-targeting siRNA (NT), or left untransfected, and were analysed after 48 h. (A) Effects of GCN2 silencing on eIF2α phosphorylation in response to UV light (UV) were analysed by western blotting. Efficiency of siRNA-mediated knockdown was monitored by staining for total and phosphorylated GCN2. Staining for actin was used as loading control. (B) Immunofluorescence microscopy staining of control or UV-treated (+UV, 20 mJ/cm2) cells incubated with or without ISRIB for 2 h post-UV exposure. SG formation was analysed by staining for G3BP1 (red) and TIAR (green). Nuclei were stained with Hoechst dye (blue). (C) UV-induced SG formation was quantified (mean±s.d.) from immunofluorescence microscopy staining represented in panel B (n=3). *P<0.05; ***P<0.001 (two-way ANOVA followed by Tukey's multiple comparisons test). (D) Concentration-dependent effects of A-92 on GCN2 and eIF2α phosphorylation in response to 20 mJ/cm2 UV light (+UV) were analysed by western blotting. Lysates from untreated cells and cells treated with sodium arsenite (+As) were used as controls. Staining for actin was used as a loading control. (E) Immunofluorescence microscopy staining of control (−) or UV-treated (UV, 20 mJ/cm2) cells incubated with 5 µM A-92 or vehicle (DMSO) for 2 h post-exposure. SG formation was visualised by staining for G3BP1 (red) and TIAR (green). Nuclei were stained with Hoechst dye (blue). (F) SG formation in control or A-92 treated cells was quantified (mean±s.d.) from immunofluorescence microscopy staining (n=3) of cells exposed to 20 mJ/cm2 UV. **P<0.01 (two-tailed Student's t-test).

Fig 3: UV-induced SG formation is dependent on G3BP1. (A–D) G3BP1, G3BP2, and control PKR knockout cells were generated using CRISPR/Cas9. (A) PKR, G3BP1 and G3BP2 expression in CRISPR knockout cells and eIF2α phosphorylation in response to 20 mJ/cm2 UV light (UV) were analysed by western blotting. Staining for actin was used as loading control. (B) UV-induced SG formation (20 mJ/cm2) was analysed by immunofluorescence microscopy in parental U2OS cells (U2OS) and CRISPR knockout cell lines lacking G3BP1 (ΔG3BP1), G3BP2 (ΔG3BP2) or PKR (ΔPKR), and stained for G3BP1 (red) and TIAR (green). Nuclei were stained with Hoechst dye (blue). (C) UV-induced SG formation was quantified (mean±s.d.) from immunofluorescence microscopy staining represented in B (n=3). *P<0.05 (two-way ANOVA followed by Tukey's multiple comparisons test). (D) Selenite-induced SG formation was analysed by immunofluorescence microscopy in parental U2OS cells (U2OS) and CRISPR knockout cell lines lacking G3BP1 (ΔG3BP1) or G3BP2 (ΔG3BP2) stained for G3BP1 (red) and G3BP2 (green). Nuclei were stained with Hoechst dye (blue). (E,F) Expression of the EGFP–USP10(1-40) fusion protein or EGFP control was induced in U2OS cells stably transduced with lentiviral constructs encoding these proteins. After 48 h, cells were left untreated (−), or were treated with UV light (UV, 20 mJ/cm2) or sodium arsenite (As). (E) Phosphorylation status of GCN2 and eIF2α post-treatment was analysed by western blotting, as well as the expression of EGFP–USP10(1-40) fusion protein or EGFP control. Staining for actin was used as loading control. (F) SG formation was analysed by immunofluorescence microscopy staining for G3BP1 (red) and TIAR (blue) in these GFP-positive cells (green). Numbers indicate the percentage of cells with SGs quantified from at least three random fields of view containing >100 cells (mean values, n=2).

Fig 4: UV-induced SG formation is decreased in GCN2-deficient cells. SG formation was compared in parental U2OS cells (WT) and GCN2-knockout cells generated using CRISPR/Cas9 (GCN2 KO). (A) SG formation was analysed by immunofluorescence microscopy in cells exposed to 20 mJ/cm2 UV light at 2 and 6 h post-exposure (UV), untreated control cells and cells treated sodium arsenite (As), and stained for G3BP1 (red) and TIAR (green). Nuclei were stained with Hoechst dye (blue). Asterisks denote cells that underwent mitosis within the time frame of the experiment (pairs of cells with predominantly cytoplasmic TIAR). (B) UV-induced SG formation in parental U2OS cells and GCN2 knockout cells was quantified at 2 h post-UV exposure from immunofluorescence microscopy staining (mean±s.d.; n=3). *P<0.05 (two-tailed Student's t-test). (C) Lack of GCN2 expression in knockout cells and its effect on eIF2α phosphorylation in response to UV light (UV) or sodium arsenite (As) were analysed by western blotting. Staining for actin was used as loading control. (D) Immunofluorescence microscopy of control (−), UV-treated (UV, 20 mJ/cm2), or arsenite-treated (As) cells stained for total GCN2 (red) and G3BP1 (green). Nuclei were stained with Hoechst dye (blue). (E) Immunofluorescence microscopy of cells treated as in D and stained for phospho-GCN2 (red) and G3BP1 (green). Nuclei were stained with Hoechst dye (blue).

Fig 5: Translation arrest and SG formation in response to UV damage is independent of mTOR inhibition. (A,B) U2OS cells were exposed to the indicated doses of UV light and incubated for 2 h with or without Torin-1 prior to analysis. (A) Phosphorylation of GCN2, eIF2α, and the mTOR substrates 4E-BP and S6 were analysed by western blotting. Lysates from cells treated with 0.5 mM sodium arsenite (As) were included for comparison. Staining for actin was used as loading control. (B) Protein synthesis rates were analysed through the ribopuromycylation assay and by western blotting with anti-puromycin antibody (Puro, top panel). Total protein content in each lane was visualised using Stain-Free reagent (bottom panel). (C) Translation arrest was quantified (mean±s.d.) from ribopuromycylation assays represented in B (n=3). *P<0.05 (one-way ANOVA followed by Tukey's multiple comparisons test). (D,E) UV-induced SG formation (20 mJ/cm2) was analysed in control cells or cells incubated in amino acid-free medium (AA-) or Torin-1-containing medium. (D) SG formation was quantified (mean±s.d.) in cells based on immunofluorescence staining (n=3). *P<0.05, **P<0.01 (two-way ANOVA followed by Tukey's multiple comparisons test). (E) Representative immunofluorescence staining for G3BP1 (red) and TIAR (green). Nuclei were stained with Hoechst dye (blue). (F) eIF4E–eIF4G and eIF4E–4E-BP interactions in cells treated with UV light (UV), amino acid-free medium (AA-), or medium with Torin-1 were analysed by means of a m7GTP-agarose pulldown assay and western blotting. The phosphorylation status of eIF2α and S6 ribosomal protein in whole-cell lysates (5% input) were determined by staining with specific antibodies. Staining for eIF4E was used as positive control, and for actin as negative controls for m7GTP pulldown.