Figure 3.
PKR transmits lysosomal damage signals leading to SG formation. (A) Unique PKR peptides and intensity (DIA); LysoIP, n = 3 (see Table S1, Tab 1). Mann-Whitney U test (LLOMe treatment relative to Ctrl). (B) Immunoblot analysis of the phosphorylation of eIF2α (S51) in Huh7 cells transfected with scrambled siRNA as control (SCR) or HRI, PKR, PERK, and GCN2 siRNA for knockdown (KD). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) was quantified based on three independent experiments. (C) Immunoblot analysis of PKR (T446) and eIF2α (S51) phosphorylation in U2OS cells treated with or without PKR inhibitor 2-AP for 1 h followed by 2 mM LLOMe treatment for 30 min as indicated. (D) Quantification by HCM of G3BP1 puncta in Huh7 cells transfected with scrambled siRNA as control (SCR) or HRI, PKR, PERK, and GCN2 siRNA for knockdown (KD). Cells were treated with 2 mM LLOMe for 30 min. Red masks, computer-identified G3BP1 puncta. (E) Quantification by HCM of G3BP1 puncta in U2OS cells treated with or without PKR inhibitor 2-AP for 1 h followed by 2 mM LLOMe treatment for 30 min as indicated. (F) Quantification by HCM of G3BP1 puncta in U2OS cells treated with or without 210 nM imidazolo-oxindole C16 for 2 h followed by 2 mM LLOMe for 30 min. Red masks, computer-identified G3BP1 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; **, P < 0.01, ANOVA. Source data are available for this figure: SourceData F3.

PKR transmits lysosomal damage signals leading to SG formation. (A) Unique PKR peptides and intensity (DIA); LysoIP, n = 3 (see Table S1, Tab 1). Mann-Whitney U test (LLOMe treatment relative to Ctrl). (B) Immunoblot analysis of the phosphorylation of eIF2α (S51) in Huh7 cells transfected with scrambled siRNA as control (SCR) or HRI, PKR, PERK, and GCN2 siRNA for knockdown (KD). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) was quantified based on three independent experiments. (C) Immunoblot analysis of PKR (T446) and eIF2α (S51) phosphorylation in U2OS cells treated with or without PKR inhibitor 2-AP for 1 h followed by 2 mM LLOMe treatment for 30 min as indicated. (D) Quantification by HCM of G3BP1 puncta in Huh7 cells transfected with scrambled siRNA as control (SCR) or HRI, PKR, PERK, and GCN2 siRNA for knockdown (KD). Cells were treated with 2 mM LLOMe for 30 min. Red masks, computer-identified G3BP1 puncta. (E) Quantification by HCM of G3BP1 puncta in U2OS cells treated with or without PKR inhibitor 2-AP for 1 h followed by 2 mM LLOMe treatment for 30 min as indicated. (F) Quantification by HCM of G3BP1 puncta in U2OS cells treated with or without 210 nM imidazolo-oxindole C16 for 2 h followed by 2 mM LLOMe for 30 min. Red masks, computer-identified G3BP1 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; **, P < 0.01, ANOVA. Source data are available for this figure: SourceData F3.

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