RNA lariats interact with G3BP protein, impairing SG function and G3BP protein stability. (A) Extracted RNA from HEK293T cells transduced with shDBR1/ID1/DKK1-lariats or control cells was used to transfect HeLa cells transduced with the scrambled shRNA or shDBR1 for 24 h; fluorescence imaging was then performed for the indicated markers. Scale bar, 20 µm. Data shown are representative of three independent experiments. (B) Lariat imaging with MCP-mScarlet and puncta quantification in HEK293T cells (n = 12 for each group). Scale bar, 20 µm. Data 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. *, P < 0.05; ****, P < 0.0001. (C) WB with the indicated antibodies following immunoprecipitation with the anti-FLAG (MCP) antibody on lysates from HEK239T cells in an overexpression system. Data shown are representative of three independent experiments. (D) HEK293T cells with ID1/DKK1-MS2-Lariats/MCP-mScarlet/G3BP-mNeonGreen/PKR-BFP/shDBR1 overexpression were imaged with the indicated fluorescence markers. Scale bar, 20 µm. Data shown are representative of three independent experiments. (E) HEK293T G3BP DKO cells with ID1/DKK1-MS2-Lariats/MCP-mScarlet/PKR-GFP/shDBR1 overexpression were imaged with indicated fluorescence markers. Scale bar, 20 µm. Data shown are representative of three independent experiments. (F and G) FRAP assay to detect the fluorescence recovery of G3BP2-mNeonGreen 3 days after shRNA transduction. Scale bars, 5 µm (F). Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. (H) HEK293T cells transduced with shDBR1 or shDBR1/ID1-DKK1-lariat were treated with MG132 or BafA1, and WB was then performed to detect the indicated proteins, followed by relative G3BP1 quantification of three independent experiments. VCL was used as a loading control. Data shown are representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. (I) MG132 was incubated for 4 h with HEK293T G3BP2-mNeonGreen cells transduced with the scrambled shRNA or shDBR1. Fluorescence imaging was then performed for the indicated markers. Scale bar, 20 µm. Data shown are representative of three independent experiments. (J) FACS detection of G3BP2-mNeonGreen protein before and after treatment with MG132. Data 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 < 0.05; ***, P < 0.001; ****, P < 0.0001. (K) MG132 or CQ was incubated with HEK293T G3BP2-mNeonGreen cells transduced with the scrambled shRNA or shDBR1, and HEK293T G3BP2-mNeonGreen cells. VCL (H and K) was used as a loading control. Data shown are representative of three independent experiments. (L) WB with the indicated antibodies following MG132 treatment and immunoprecipitation with an anti-FLAG (MCP) antibody on lysates of HEK293T cells in a stable overexpression system. Data shown are representative of three independent experiments. Source data are available for this figure: SourceData F6.
Figure 6.

RNA lariats interact with G3BP protein, impairing SG function and G3BP protein stability. (A) Extracted RNA from HEK293T cells transduced with shDBR1/ID1/DKK1-lariats or control cells was used to transfect HeLa cells transduced with the scrambled shRNA or shDBR1 for 24 h; fluorescence imaging was then performed for the indicated markers. Scale bar, 20 µm. Data shown are representative of three independent experiments. (B) Lariat imaging with MCP-mScarlet and puncta quantification in HEK293T cells (n = 12 for each group). Scale bar, 20 µm. Data 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. *, P < 0.05; ****, P < 0.0001. (C) WB with the indicated antibodies following immunoprecipitation with the anti-FLAG (MCP) antibody on lysates from HEK239T cells in an overexpression system. Data shown are representative of three independent experiments. (D) HEK293T cells with ID1/DKK1-MS2-Lariats/MCP-mScarlet/G3BP-mNeonGreen/PKR-BFP/shDBR1 overexpression were imaged with the indicated fluorescence markers. Scale bar, 20 µm. Data shown are representative of three independent experiments. (E) HEK293T G3BP DKO cells with ID1/DKK1-MS2-Lariats/MCP-mScarlet/PKR-GFP/shDBR1 overexpression were imaged with indicated fluorescence markers. Scale bar, 20 µm. Data shown are representative of three independent experiments. (F and G) FRAP assay to detect the fluorescence recovery of G3BP2-mNeonGreen 3 days after shRNA transduction. Scale bars, 5 µm (F). Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. (H) HEK293T cells transduced with shDBR1 or shDBR1/ID1-DKK1-lariat were treated with MG132 or BafA1, and WB was then performed to detect the indicated proteins, followed by relative G3BP1 quantification of three independent experiments. VCL was used as a loading control. Data shown are representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. (I) MG132 was incubated for 4 h with HEK293T G3BP2-mNeonGreen cells transduced with the scrambled shRNA or shDBR1. Fluorescence imaging was then performed for the indicated markers. Scale bar, 20 µm. Data shown are representative of three independent experiments. (J) FACS detection of G3BP2-mNeonGreen protein before and after treatment with MG132. Data 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 < 0.05; ***, P < 0.001; ****, P < 0.0001. (K) MG132 or CQ was incubated with HEK293T G3BP2-mNeonGreen cells transduced with the scrambled shRNA or shDBR1, and HEK293T G3BP2-mNeonGreen cells. VCL (H and K) was used as a loading control. Data shown are representative of three independent experiments. (L) WB with the indicated antibodies following MG132 treatment and immunoprecipitation with an anti-FLAG (MCP) antibody on lysates of HEK293T cells in a stable overexpression system. Data shown are representative of three independent experiments. Source data are available for this figure: SourceData F6.

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