Figure S3.
The inhibitory effect of PCK1 S151 phosphorylation on IFN-I depends on cGAS-STING pathway. (A) Parental and cGAS-KO BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or S151A mutant treated with or without hypoxia for 12 h were harvested for bulk RNA-seq analyses. Principal component analysis of the variance-stabilized estimated raw counts of differentially expressed genes in the cells from six groups as indicated (n = 3). (B) Parental or STING-KO BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants treated with or without hypoxia for 6 h were harvested for immunoblotting analyses as indicated. (C) Parental or STING-KO BT-549 and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants, which 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) Parental or STING-KO MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 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). The experiment (B) was 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 (C and D); *P < 0.05, **P < 0.001, and ***P < 0.0001; NS, not significant. Source data are available for this figure: SourceData FS3. Refer to the image caption for details.

The inhibitory effect of PCK1 S151 phosphorylation on IFN-I depends on cGAS-STING pathway. (A) Parental and cGAS-KO BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or S151A mutant treated with or without hypoxia for 12 h were harvested for bulk RNA-seq analyses. Principal component analysis of the variance-stabilized estimated raw counts of differentially expressed genes in the cells from six groups as indicated (n = 3). (B) Parental or STING-KO BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants treated with or without hypoxia for 6 h were harvested for immunoblotting analyses as indicated. (C) Parental or STING-KO BT-549 and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants, which 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) Parental or STING-KO MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 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). The experiment (B) was 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 (C and D); *P < 0.05, **P < 0.001, and ***P < 0.0001; NS, not significant. Source data are available for this figure: SourceData FS3.

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