Figure 8. PCK1 S151... | Rockefeller University Press

Figure 8.

PCK1 S151 phosphorylation–mediated cGAS inhibition promotes breast tumor growth. (A–C) Parental or cGAS-KO 4T-1 cells (1 × 106) expressing mouse PCK1 shRNA with reconstituted expression of Flag-rmPCK1 WT or S151A mutant were injected into the mammary fat pad of female BALB/c mice. The mice were euthanized and examined for tumor growth 22 days after injection (A), tumor volumes were calculated (B), and tumors were weighed (C) (n = 6 per group). (D and E) IHC analyses of the indicated xenograft tumor samples in A were performed with the indicated antibodies. Representative staining images are shown (D), and the staining scores for the indicated tumor samples were compared (E). Scale bar, 50 μm. The indicated staining scores were quantified in n = 18 microscopic fields. (F and G) Representative bioluminescence images from the indicated days after the orthotopical injection of EMT-6 luciferase-expressing cells (1 × 106) expressing mouse PCK1 shRNA with reconstituted expression of Flag-rmPCK1 WT or S151A mutant into the mammary fat pad of female BALB/c mice (F). The quantification of the bioluminescence imaging is shown (G) (n = 6 per group). (H and I) IHC analyses of the indicated xenograft tumor samples in F were performed with the indicated antibodies. Representative staining images are shown (H), and staining scores for the indicated tumor samples were compared (I). Scale bar, 50 μm. The indicated staining scores were quantified in n = 18 microscopic fields. (J and K) 4T-1 cells (1 × 106) expressing mouse PCK1 shRNA with reconstituted expression of Flag-rmPCK1 WT or S151A mutant were injected into the mammary fat pad of female BALB/c mice. Mice were put in an ambient atmosphere (21% O2) or 11% oxygen tension for 30 days after injection, tumor volumes were calculated (J), and tumors were weighed (K) (n = 6 per group). (L and M) IHC analyses of the indicated xenograft tumor samples in J were performed with the indicated antibodies. Representative staining images are shown (L), and the indicated staining scores were quantified in n = 18 microscopic fields (M). Scale bar, 50 μm. Data are representative of as mean ± SD. Statistical significance was determined by two-tailed Student’s t test (C, E, G, I, K, and M) and two-way ANOVA followed by Tukey’s test (B and J); *P < 0.05, **P < 0.001, and ***P < 0.0001; NS, not significant. Refer to the image caption for details.

PCK1 S151 phosphorylation–mediated cGAS inhibition promotes breast tumor growth. (A–C) Parental or cGAS-KO 4T-1 cells (1 × 10⁶) expressing mouse PCK1 shRNA with reconstituted expression of Flag-rmPCK1 WT or S151A mutant were injected into the mammary fat pad of female BALB/c mice. The mice were euthanized and examined for tumor growth 22 days after injection (A), tumor volumes were calculated (B), and tumors were weighed (C) (n = 6 per group). (D and E) IHC analyses of the indicated xenograft tumor samples in A were performed with the indicated antibodies. Representative staining images are shown (D), and the staining scores for the indicated tumor samples were compared (E). Scale bar, 50 μm. The indicated staining scores were quantified in n = 18 microscopic fields. (F and G) Representative bioluminescence images from the indicated days after the orthotopical injection of EMT-6 luciferase-expressing cells (1 × 10⁶) expressing mouse PCK1 shRNA with reconstituted expression of Flag-rmPCK1 WT or S151A mutant into the mammary fat pad of female BALB/c mice (F). The quantification of the bioluminescence imaging is shown (G) (n = 6 per group). (H and I) IHC analyses of the indicated xenograft tumor samples in F were performed with the indicated antibodies. Representative staining images are shown (H), and staining scores for the indicated tumor samples were compared (I). Scale bar, 50 μm. The indicated staining scores were quantified in n = 18 microscopic fields. (J and K) 4T-1 cells (1 × 10⁶) expressing mouse PCK1 shRNA with reconstituted expression of Flag-rmPCK1 WT or S151A mutant were injected into the mammary fat pad of female BALB/c mice. Mice were put in an ambient atmosphere (21% O₂) or 11% oxygen tension for 30 days after injection, tumor volumes were calculated (J), and tumors were weighed (K) (n = 6 per group). (L and M) IHC analyses of the indicated xenograft tumor samples in J were performed with the indicated antibodies. Representative staining images are shown (L), and the indicated staining scores were quantified in n = 18 microscopic fields (M). Scale bar, 50 μm. Data are representative of as mean ± SD. Statistical significance was determined by two-tailed Student’s t test (C, E, G, I, K, and M) and two-way ANOVA followed by Tukey’s test (B and J); *P < 0.05, **P < 0.001, and ***P < 0.0001; NS, not significant.