Figure S2.
PCK1 S151 phosphorylation attenuates hypoxia-mediated activation of cGAS-STING pathway. (A) Parental MDA-MB-231 cells and the indicated clones with knock-in expression of PCK1 S151A mutants 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). (B) Genomic DNA was extracted from two individual clones of parental 4T-1 cells with knock-in expression of PCK1 S151A. PCR products amplified from the indicated DNA fragments were separated on an agarose gel. (C) Sequencing of parental 4T-1 cells and the individual clones of parental cells with knock-in expression of PCK1 S151A. The red line indicates the sgRNA-targeting sequence. The black line indicates the protospacer adjacent motif (PAM). Blue arrows indicate mutated nucleotides. A mutated amino acid and its WT counterpart are indicated by the solid red box. (D) Parental 4T-1 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 4T-1 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). (F) Parental 4T-1 cells and the indicated clones with knock-in expression of PCK1 S151A mutants 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). Experiments (D) were repeated three times independently with similar results, and the representative data are shown. Data are representative of as mean ± SD. Statistical significance was determined by two-tailed Student’s t test (A, E, and F); *P < 0.05 and ***P < 0.0001. Source data are available for this figure: SourceData FS2. Refer to the image caption for details.

PCK1 S151 phosphorylation attenuates hypoxia-mediated activation of cGAS-STING pathway. (A) Parental MDA-MB-231 cells and the indicated clones with knock-in expression of PCK1 S151A mutants 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). (B) Genomic DNA was extracted from two individual clones of parental 4T-1 cells with knock-in expression of PCK1 S151A. PCR products amplified from the indicated DNA fragments were separated on an agarose gel. (C) Sequencing of parental 4T-1 cells and the individual clones of parental cells with knock-in expression of PCK1 S151A. The red line indicates the sgRNA-targeting sequence. The black line indicates the protospacer adjacent motif (PAM). Blue arrows indicate mutated nucleotides. A mutated amino acid and its WT counterpart are indicated by the solid red box. (D) Parental 4T-1 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 4T-1 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). (F) Parental 4T-1 cells and the indicated clones with knock-in expression of PCK1 S151A mutants 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). Experiments (D) were repeated three times independently with similar results, and the representative data are shown. Data are representative of as mean ± SD. Statistical significance was determined by two-tailed Student’s t test (A, E, and F); *P < 0.05 and ***P < 0.0001. Source data are available for this figure: SourceData FS2.

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