Figure 7.
The GTP-binding affinity of PCK1 and cGAS is indispensable for PCK1 S151 phosphorylation–mediated cGAS-STING inhibition. (A) BLI was used to test the interaction of GTP to recombinant His-cGAS WT or His-cGAS T321L. Sensors were loaded with biotinylated His-cGAS WT or His-cGAS T321L proteins. (B) MDA-MB-231 cells expressing PCK1 shRNA and cGAS shRNA with reconstituted expression of the indicated PCK1 or cGAS proteins treated with or without hypoxia for 6 h were harvested for immunoblotting analyses as indicated. (C) MDA-MB-231 cells expressing PCK1 shRNA and cGAS shRNA with reconstituted expression of the indicated PCK1 or cGAS proteins were transiently transfected with vectors expressing β-galactosidase and an IFN-β luciferase reporter. 24 h later, the aforementioned cells treated with or without hypoxia for 6 h were harvested for luciferase assay, and the relative IFN-β luciferase activity after normalization to β-galactosidase activity is shown (n = 4 biological experiments). (D) MDA-MB-231 cells expressing PCK1 shRNA and cGAS shRNA with reconstituted expression of the indicated PCK1 or cGAS proteins treated with or without hypoxia for 12 h were harvested for qPCR analyses to measure CXCL10, IL6, IFNB1, and IFNA4 mRNA levels (n = 6 biological experiments). (E) BLI was used to test the interaction of GTP to recombinant GST-PCK1 WT or GST-PCK1 N533A, A287V, or R436A mutants. Sensors were loaded with biotinylated GST-PCK1 WT or GST-PCK1 N533A, A287V, or R436A proteins. (F) MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of the indicated PCK1 proteins transfected with or without 2 μg/ml HT-DNA for 6 h were harvested for immunoblotting analyses as indicated. (G) MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants were transiently transfected with vectors expressing β-galactosidase and an IFN-β luciferase reporter. 24 h later, the aforementioned cells treated with or without 2 μg/ml HT-DNA for 6 h were harvested for luciferase assay, and the relative IFN-β luciferase activity after normalization to β-galactosidase activity is shown (n = 4 biological experiments). (H) MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants treated with or without 2 μg/ml HT-DNA for 12 h were harvested for qPCR analyses to measure CXCL10, IL6, IFNB1, and IFNA4 mRNA levels (n = 6 biological experiments). Experiments (A, B, E, and F) were repeated three times independently with similar results, and the representative data are shown. Statistical significance was determined by two-tailed Student’s t test (C, D, G, and H). Data are the mean ± SD; *P < 0.05, **P < 0.001, and ***P < 0.0001; NS, not significant. Source data are available for this figure: SourceData F7. Refer to the image caption for details.

The GTP-binding affinity of PCK1 and cGAS is indispensable for PCK1 S151 phosphorylation–mediated cGAS-STING inhibition. (A) BLI was used to test the interaction of GTP to recombinant His-cGAS WT or His-cGAS T321L. Sensors were loaded with biotinylated His-cGAS WT or His-cGAS T321L proteins. (B) MDA-MB-231 cells expressing PCK1 shRNA and cGAS shRNA with reconstituted expression of the indicated PCK1 or cGAS proteins treated with or without hypoxia for 6 h were harvested for immunoblotting analyses as indicated. (C) MDA-MB-231 cells expressing PCK1 shRNA and cGAS shRNA with reconstituted expression of the indicated PCK1 or cGAS proteins were transiently transfected with vectors expressing β-galactosidase and an IFN-β luciferase reporter. 24 h later, the aforementioned cells treated with or without hypoxia for 6 h were harvested for luciferase assay, and the relative IFN-β luciferase activity after normalization to β-galactosidase activity is shown (n = 4 biological experiments). (D) MDA-MB-231 cells expressing PCK1 shRNA and cGAS shRNA with reconstituted expression of the indicated PCK1 or cGAS proteins treated with or without hypoxia for 12 h were harvested for qPCR analyses to measure CXCL10, IL6, IFNB1, and IFNA4 mRNA levels (n = 6 biological experiments). (E) BLI was used to test the interaction of GTP to recombinant GST-PCK1 WT or GST-PCK1 N533A, A287V, or R436A mutants. Sensors were loaded with biotinylated GST-PCK1 WT or GST-PCK1 N533A, A287V, or R436A proteins. (F) MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of the indicated PCK1 proteins transfected with or without 2 μg/ml HT-DNA for 6 h were harvested for immunoblotting analyses as indicated. (G) MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants were transiently transfected with vectors expressing β-galactosidase and an IFN-β luciferase reporter. 24 h later, the aforementioned cells treated with or without 2 μg/ml HT-DNA for 6 h were harvested for luciferase assay, and the relative IFN-β luciferase activity after normalization to β-galactosidase activity is shown (n = 4 biological experiments). (H) MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants treated with or without 2 μg/ml HT-DNA for 12 h were harvested for qPCR analyses to measure CXCL10, IL6, IFNB1, and IFNA4 mRNA levels (n = 6 biological experiments). Experiments (A, B, E, and F) were repeated three times independently with similar results, and the representative data are shown. Statistical significance was determined by two-tailed Student’s t test (C, D, G, and H). Data are the mean ± SD; *P < 0.05, **P < 0.001, and ***P < 0.0001; NS, not significant. Source data are available for this figure: SourceData F7.

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