Figure 2.
PCK1 S151 phosphorylation attenuates hypoxia-mediated activation of cGAS-STING pathway. (A) Parental BT-549 cells and the indicated clones with knock-in expression of PCK1 S151A mutants treated with or without hypoxia for 6 h were prepared for IF analyses as indicated (left). Scale bar, 20 μm. The co-localization coefficients between the indicated proteins in the presence or absence of hypoxia are shown (right). At least 10 cells from each independent experiment were analyzed, and the representative data are shown (n = 6 biological experiments). (B) Parental BT-549 and MDA-MB-231 cells and the indicated clones with knock-in expression of PCK1 S151A mutants treated with or without hypoxia for 6 h were resolved by native PAGE or SDS-PAGE, followed by immunoblotting analysis as indicated. (C) Parental BT-549 and MDA-MB-231 cells and the indicated clones with knock-in expression of PCK1 S151A mutants transfected with HA-K63-UB and treated with or without hypoxia for 6 h were harvested for IP and immunoblotting analyses as indicated. (D) Parental BT-549 and MDA-MB-231 cells and the indicated clones with knock-in expression of PCK1 S151A mutants treated with or without hypoxia for 6 h were harvested for immunoblotting analyses as indicated. (E) Parental BT-549 cells and the indicated clones with knock-in expression of PCK1 S151A mutants treated with or without hypoxia for 6 h were prepared for IF analyses as indicated (upper). Scale bar, 20 μm. The co-localization coefficients between the indicated proteins in the presence or absence of hypoxia are shown (lower). At least 10 cells from each independent experiment were analyzed, and the representative data are shown (n = 6 biological experiments). (F) Parental BT-549 (left) and MDA-MB-231 (right) cells and the indicated clones with knock-in expression of PCK1 S151A 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 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). (G) Parental BT-549 cells and the indicated clones with knock-in expression of PCK1 S151A mutants treated with or without hypoxia for 12 h were harvested for quantitative PCR (qPCR) analyses to measure CXCL10, IL6, IFNB1, and IFNA4 mRNA levels (n = 6 biological experiments). Experiments (B, C, and D) 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 (A and E–G). Data are the mean ± SD; *P < 0.05, **P < 0.001, and ***P < 0.0001. Source data are available for this figure: SourceData F2. Refer to the image caption for details.

PCK1 S151 phosphorylation attenuates hypoxia-mediated activation of cGAS-STING pathway. (A) Parental BT-549 cells and the indicated clones with knock-in expression of PCK1 S151A mutants treated with or without hypoxia for 6 h were prepared for IF analyses as indicated (left). Scale bar, 20 μm. The co-localization coefficients between the indicated proteins in the presence or absence of hypoxia are shown (right). At least 10 cells from each independent experiment were analyzed, and the representative data are shown (n = 6 biological experiments). (B) Parental BT-549 and MDA-MB-231 cells and the indicated clones with knock-in expression of PCK1 S151A mutants treated with or without hypoxia for 6 h were resolved by native PAGE or SDS-PAGE, followed by immunoblotting analysis as indicated. (C) Parental BT-549 and MDA-MB-231 cells and the indicated clones with knock-in expression of PCK1 S151A mutants transfected with HA-K63-UB and treated with or without hypoxia for 6 h were harvested for IP and immunoblotting analyses as indicated. (D) Parental BT-549 and MDA-MB-231 cells and the indicated clones with knock-in expression of PCK1 S151A mutants treated with or without hypoxia for 6 h were harvested for immunoblotting analyses as indicated. (E) Parental BT-549 cells and the indicated clones with knock-in expression of PCK1 S151A mutants treated with or without hypoxia for 6 h were prepared for IF analyses as indicated (upper). Scale bar, 20 μm. The co-localization coefficients between the indicated proteins in the presence or absence of hypoxia are shown (lower). At least 10 cells from each independent experiment were analyzed, and the representative data are shown (n = 6 biological experiments). (F) Parental BT-549 (left) and MDA-MB-231 (right) cells and the indicated clones with knock-in expression of PCK1 S151A 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 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). (G) Parental BT-549 cells and the indicated clones with knock-in expression of PCK1 S151A mutants treated with or without hypoxia for 12 h were harvested for quantitative PCR (qPCR) analyses to measure CXCL10, IL6, IFNB1, and IFNA4 mRNA levels (n = 6 biological experiments). Experiments (B, C, and D) 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 (A and E–G). Data are the mean ± SD; *P < 0.05, **P < 0.001, and ***P < 0.0001. Source data are available for this figure: SourceData F2.

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