G3BP SG assembly and G3BP protein levels remain low in DBR1-deficient cells. (A) BJ cells transduced with the scrambled control shRNA or with shDBR1 were infected with the indicated viruses, and SG formation was then followed by G3BP1 staining, microscopy imaging, and the SG puncta quantification. Scale bar, 20 µm. Data shown are representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ****, P < 0.0001. (B) HEK293T cells transduced with the scrambled shRNA or with shDBR1 were stimulated with polyI:C (1 µg/ml) or infected with VSV (MOI: 0.01) or HSV-1 (MOI: 0.05) and G3BP1 was then detected by fluorescence. Scale bar, 20 µm. Data shown are representative of six independent experiments. (C) HeLa cells transduced with the scrambled control shRNA or with shDBR1 were stimulated with polyI:C (1 µg/ml) and G3BP1 was then detected by fluorescence microscopy. Scale bar, 20 µm. Data shown are representative of three independent experiments. (D and E) Control cells and cells from a DBR1-deficient patient were infected with VSV (MOI = 0.01) for 24 h, stimulated with polyI:C (1 µg/ml) for 4 h (D), or treated with 500 µM sodium arsenite (SA) for 1 h (E). Immunofluorescence staining was then performed for the indicated markers and SGs were quantified. Scale bar, 20 µm. Data shown are representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant, **, P < 0.01; ****, P < 0.0001. (F) HEK293T cells transduced with shDBR1 or DBR1-mscarlet were subjected to immunofluorescence staining for the indicated markers. Scale bar, 20 µm. Data shown are representative of three independent experiments. (G) HEK293T cells transduced with the scrambled control shRNA, and shDBR1 cells expressing G3BP2-mNeonGreen were transfected with polyI:C (1 µg/ml) for 4 h, and the markers indicated were then detected by fluorescence microscopy. Scale bar, 20 µm. Data shown are representative of six independent experiments. (H) RT-qPCR and WB detection of G3BP expression in indicated HeLa cells. GAPDH was used as a loading control. Data shown are representative of three independent experiments. Graph points represent biological replicates. (I) FACS analysis of the expression of G3BP (uninfected) and viral GFP after VSV-GFP (MOI: 0.01) infection for 24 h in EBV-B cells from a control or a DBR1-deficient patient. Data shown are representative of three independent experiments. (J and K) G3BP1+/+ and G3BP1−/− HeLa cells transduced with EV or DBR1 were infected with VSV-GFP (MOI: 0.01); viral gene expression was then quantified by RT-qPCR (J). Data shown are representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01; ****, P < 0.0001. Proteins were detected by WB detection with indicated antibodies (K). Vinculin (VCL) was used as a loading control. Data shown are representative of three independent experiments. (L) Scrambled shRNA and shDBR1 HeLa cells transduced with EV or G3BP1 were infected with HSV-1. Viral gene expression was then quantified by RT-qPCR. Data shown are representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant, ***, P < 0.001; ****, P < 0.0001. Source data are available for this figure: SourceData F5.
Figure 5.

G3BP SG assembly and G3BP protein levels remain low in DBR1-deficient cells. (A) BJ cells transduced with the scrambled control shRNA or with shDBR1 were infected with the indicated viruses, and SG formation was then followed by G3BP1 staining, microscopy imaging, and the SG puncta quantification. Scale bar, 20 µm. Data shown are representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ****, P < 0.0001. (B) HEK293T cells transduced with the scrambled shRNA or with shDBR1 were stimulated with polyI:C (1 µg/ml) or infected with VSV (MOI: 0.01) or HSV-1 (MOI: 0.05) and G3BP1 was then detected by fluorescence. Scale bar, 20 µm. Data shown are representative of six independent experiments. (C) HeLa cells transduced with the scrambled control shRNA or with shDBR1 were stimulated with polyI:C (1 µg/ml) and G3BP1 was then detected by fluorescence microscopy. Scale bar, 20 µm. Data shown are representative of three independent experiments. (D and E) Control cells and cells from a DBR1-deficient patient were infected with VSV (MOI = 0.01) for 24 h, stimulated with polyI:C (1 µg/ml) for 4 h (D), or treated with 500 µM sodium arsenite (SA) for 1 h (E). Immunofluorescence staining was then performed for the indicated markers and SGs were quantified. Scale bar, 20 µm. Data shown are representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant, **, P < 0.01; ****, P < 0.0001. (F) HEK293T cells transduced with shDBR1 or DBR1-mscarlet were subjected to immunofluorescence staining for the indicated markers. Scale bar, 20 µm. Data shown are representative of three independent experiments. (G) HEK293T cells transduced with the scrambled control shRNA, and shDBR1 cells expressing G3BP2-mNeonGreen were transfected with polyI:C (1 µg/ml) for 4 h, and the markers indicated were then detected by fluorescence microscopy. Scale bar, 20 µm. Data shown are representative of six independent experiments. (H) RT-qPCR and WB detection of G3BP expression in indicated HeLa cells. GAPDH was used as a loading control. Data shown are representative of three independent experiments. Graph points represent biological replicates. (I) FACS analysis of the expression of G3BP (uninfected) and viral GFP after VSV-GFP (MOI: 0.01) infection for 24 h in EBV-B cells from a control or a DBR1-deficient patient. Data shown are representative of three independent experiments. (J and K) G3BP1+/+ and G3BP1−/− HeLa cells transduced with EV or DBR1 were infected with VSV-GFP (MOI: 0.01); viral gene expression was then quantified by RT-qPCR (J). Data shown are representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01; ****, P < 0.0001. Proteins were detected by WB detection with indicated antibodies (K). Vinculin (VCL) was used as a loading control. Data shown are representative of three independent experiments. (L) Scrambled shRNA and shDBR1 HeLa cells transduced with EV or G3BP1 were infected with HSV-1. Viral gene expression was then quantified by RT-qPCR. Data shown are representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant, ***, P < 0.001; ****, P < 0.0001. Source data are available for this figure: SourceData F5.

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