Figure S4.
The GTP-binding affinity of PCK1 and cGAS is indispensable for PCK1 S151 phosphorylation–mediated cGAS-STING inhibition. (A and B) Enzymatic kinetic plot of His-cGAS and His-PCK1 toward GTP is presented (A) (n = 3 biological experiments), and Coomassie blue staining (B) was performed to show the purity of bacterially purified proteins in A. (C) MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or S151D mutant transfected with or without 2 μg/ml HT-DNA in the presence or absence of 0.5 mM ATP or GTP and digitonin (25 mg/ml) for 12 h were harvested for qPCR analyses to measure CXCL10, IL6, IFNB1, and IFNA4 mRNA levels (n = 6 biological experiments). (D) The thermal stability of His-cGAS WT or T321L mutant was detected by TSA. RFU, relative fluorescence units. (E) The purity of bacterially purified His-cGAS WT and T321L mutant was analyzed by Coomassie blue staining. (F) MDA-MB-231 cells expressing PCK1 shRNA and cGAS shRNA with reconstituted expression of the indicated PCK1 or cGAS proteins were harvested for immunoblotting analyses as indicated. (G) The thermal stability of GST-PCK1 WT or GST-PCK1 N533A, A287V, or R436A mutants was detected by TSA. RFU, relative fluorescence units. (H) The purity of bacterially purified GST-PCK1 WT or GST-PCK1 N533A, A287V, or R436A mutants was analyzed by Coomassie blue staining. (I) MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants were harvested for immunoblotting analyses as indicated. (J and K) 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 prepared for IF analyses with an anti-dsDNA–specific antibody (J, left) or 3 μl/ml PicoGreen staining (K, left). Scale bar, 20 μm. Graphical representation shows dsDNA and PicoGreen quantitation, respectively (J and K, right). At least n = 30 cells from each independent experiment were analyzed and representative data are shown. (L) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected with HA-tagged WT or DNA-binding defective mutant (K173A/R176A) were harvested for the biotin pull-down assay to measure the DNA-binding ability of cGAS. ISD indicates IFN stimulatory DNA. (M) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected with Myc-tagged WT or cGAS K394E were harvested for IP and immunoblotting analyses as indicated. Experiments (D–I, L, and M) 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 (C), two-way ANOVA, followed by Tukey’s test (J and K); **P < 0.001 and ***P < 0.0001; NS, not significant. Source data are available for this figure: SourceData FS4. Refer to the image caption for details.

The GTP-binding affinity of PCK1 and cGAS is indispensable for PCK1 S151 phosphorylation–mediated cGAS-STING inhibition. (A and B) Enzymatic kinetic plot of His-cGAS and His-PCK1 toward GTP is presented (A) (n = 3 biological experiments), and Coomassie blue staining (B) was performed to show the purity of bacterially purified proteins in A. (C) MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or S151D mutant transfected with or without 2 μg/ml HT-DNA in the presence or absence of 0.5 mM ATP or GTP and digitonin (25 mg/ml) for 12 h were harvested for qPCR analyses to measure CXCL10, IL6, IFNB1, and IFNA4 mRNA levels (n = 6 biological experiments). (D) The thermal stability of His-cGAS WT or T321L mutant was detected by TSA. RFU, relative fluorescence units. (E) The purity of bacterially purified His-cGAS WT and T321L mutant was analyzed by Coomassie blue staining. (F) MDA-MB-231 cells expressing PCK1 shRNA and cGAS shRNA with reconstituted expression of the indicated PCK1 or cGAS proteins were harvested for immunoblotting analyses as indicated. (G) The thermal stability of GST-PCK1 WT or GST-PCK1 N533A, A287V, or R436A mutants was detected by TSA. RFU, relative fluorescence units. (H) The purity of bacterially purified GST-PCK1 WT or GST-PCK1 N533A, A287V, or R436A mutants was analyzed by Coomassie blue staining. (I) MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants were harvested for immunoblotting analyses as indicated. (J and K) 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 prepared for IF analyses with an anti-dsDNA–specific antibody (J, left) or 3 μl/ml PicoGreen staining (K, left). Scale bar, 20 μm. Graphical representation shows dsDNA and PicoGreen quantitation, respectively (J and K, right). At least n = 30 cells from each independent experiment were analyzed and representative data are shown. (L) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected with HA-tagged WT or DNA-binding defective mutant (K173A/R176A) were harvested for the biotin pull-down assay to measure the DNA-binding ability of cGAS. ISD indicates IFN stimulatory DNA. (M) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected with Myc-tagged WT or cGAS K394E were harvested for IP and immunoblotting analyses as indicated. Experiments (D–I, L, and M) 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 (C), two-way ANOVA, followed by Tukey’s test (J and K); **P < 0.001 and ***P < 0.0001; NS, not significant. Source data are available for this figure: SourceData FS4.

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