Figure 3.
PCK1 suppresses cGAS-STING activation upon hypoxia in an enzymatic-dependent manner. (A) BT-549 and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or C288S mutant were harvested for immunoblotting analyses. (B) BT-549 cells and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or C288S mutant treated with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. (C) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or C288S mutant 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). (D) BT-549 cells and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or C288S mutant treated with or without hypoxia for 6 h were harvested for immunoblotting analyses as indicated. (E) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT and C288S protein 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) BT-549 cells and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT and C288S protein 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) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT and C288S protein 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 (A, B, 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 (C and E–G). Data are the mean ± SD; **P < 0.001 and ***P < 0.0001. Source data are available for this figure: SourceData F3. Refer to the image caption for details.

PCK1 suppresses cGAS-STING activation upon hypoxia in an enzymatic-dependent manner. (A) BT-549 and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or C288S mutant were harvested for immunoblotting analyses. (B) BT-549 cells and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or C288S mutant treated with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. (C) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or C288S mutant 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). (D) BT-549 cells and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or C288S mutant treated with or without hypoxia for 6 h were harvested for immunoblotting analyses as indicated. (E) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT and C288S protein 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) BT-549 cells and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT and C288S protein 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) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT and C288S protein 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 (A, B, 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 (C and E–G). Data are the mean ± SD; **P < 0.001 and ***P < 0.0001. Source data are available for this figure: SourceData F3.

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