Figure 5.
PCK1 S151 phosphorylation–mediated PCK1–cGAS interaction limits cGAS-STING activation via inhibiting cGAS activity. (A) BT-549 and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected with or without 2 μg/ml HT-DNA for 6 h were harvested for immunoblotting analyses as indicated. (B and C) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected with or without 2 μg/ml HT-DNA for 6 h were prepared for IF analyses as indicated (B, left; C, left). Scale bar, 20 μm.The co-localization coefficients between the indicated proteins in the presence or absence of HT-DNA stimulation are shown (B, right; C, right). At least 10 cells from each independent experiment were analyzed, and the representative data are shown (n = 6 biological experiments). (D) BT-549 (left) and MDA-MB-231 (right) cells expressing PCK1 shRNA with reconstituted expression of the indicated PCK1 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 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). (E) MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected 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). (F) BT-549 and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants treated with or without cGAMP for 6 h were harvested for immunoblotting analyses as indicated. (G and H) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants treated with or without cGAMP for 6 h were prepared for IF analyses as indicated (G, left; H, left). Scale bar, 20 μm.The co-localization coefficients between the indicated proteins in the presence or absence of cGAMP stimulation (G, right; H, right). At least 10 cells from each independent experiment were analyzed and representative data are shown (n = 6 biological experiments). (I) BT-549 (left) and MDA-MB-231 cells (right) 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 cGAMP 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). (J) MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of the indicated PCK1 proteins treated with or without cGAMP for 12 h were harvested for qPCR analyses to measure CXCL10, IL6, IFNB1, and IFNA4 mRNA levels (n = 6 biological experiments). Experiments (A 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 (B–E and G–J). 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 F5. Refer to the image caption for details.

PCK1 S151 phosphorylation–mediated PCK1–cGAS interaction limits cGAS-STING activation via inhibiting cGAS activity. (A) BT-549 and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected with or without 2 μg/ml HT-DNA for 6 h were harvested for immunoblotting analyses as indicated. (B and C) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected with or without 2 μg/ml HT-DNA for 6 h were prepared for IF analyses as indicated (B, left; C, left). Scale bar, 20 μm.The co-localization coefficients between the indicated proteins in the presence or absence of HT-DNA stimulation are shown (B, right; C, right). At least 10 cells from each independent experiment were analyzed, and the representative data are shown (n = 6 biological experiments). (D) BT-549 (left) and MDA-MB-231 (right) cells expressing PCK1 shRNA with reconstituted expression of the indicated PCK1 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 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). (E) MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected 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). (F) BT-549 and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants treated with or without cGAMP for 6 h were harvested for immunoblotting analyses as indicated. (G and H) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants treated with or without cGAMP for 6 h were prepared for IF analyses as indicated (G, left; H, left). Scale bar, 20 μm.The co-localization coefficients between the indicated proteins in the presence or absence of cGAMP stimulation (G, right; H, right). At least 10 cells from each independent experiment were analyzed and representative data are shown (n = 6 biological experiments). (I) BT-549 (left) and MDA-MB-231 cells (right) 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 cGAMP 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). (J) MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of the indicated PCK1 proteins treated with or without cGAMP for 12 h were harvested for qPCR analyses to measure CXCL10, IL6, IFNB1, and IFNA4 mRNA levels (n = 6 biological experiments). Experiments (A 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 (B–E and G–J). 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 F5.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal