PCK1 S151 phosphorylation attenuates hypoxia-induced IFN-I signaling in a cGAS-dependent manner. (A) Parental and cGAS-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. (B) Parental and cGAS-KO BT-549 cells (upper) and MDA-MB-231 cells (lower) 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 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). (C) Parental and cGAS-KO BT-549 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). (D) 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. Heatmap represents differentially expressed genes in the indicated groups (n = 3 biological experiments). (E and F) Parental (E) and cGAS-KO (F) BT-549 cells expressing PCK1 shRNA with reconstituted expression of the Flag-rPCK1 WT or S151A mutant treated with hypoxia for 12 h. Volcano plot showing the differentially expressed genes between the indicated groups (absolute log₂ fold change >1; P < 0.05) (n = 3 biological experiments). (F) 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. The differently expressing ISGs in the indicated groups were identified using transcriptomic data in D and presented as the heatmap plot (n = 3 biological experiments). (G) Parental (left) or cGAS KO (right) BT-549 cells expressing PCK1 shRNA with reconstituted expression of the Flag-rPCK1 WT or S151A mutant treated with hypoxia for 12 h. Functional enrichment (GO and KEGG) analyses of IFN-I–associated pathways enriched between the indicated groups were identified using transcriptomic data in D (n = 3 biological experiments). (H) Parental (left) or cGAS KO (right) BT-549 cells expressing PCK1 shRNA with reconstituted expression of the Flag-rPCK1 WT or S151A mutant treated with hypoxia for 12 h. Gene set enrichment analysis (GSEA) showing the representative IFN-I–associated pathways enriched between the indicated groups were identified using transcriptomic data in D (n = 3 biological experiments). The experiment (A) was 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 and C). 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 F4.