Fig. 2. The cGAS-STING signaling pathway plays a central role in the proinflammatory SASP induced by typhoid toxin.
a GSEA showing upregulated genes related to cytosolic DNA sensing pathway in macrophages treated with wild-type (WT) typhoid toxin compared to those treated with the PltBS35A mutant. b Representative images of immunofluorescent staining in Henle-407 cells treated with WT typhoid toxin, the PltBS35A and the CdtBH160Q mutants with anti-dsDNA and DAPI are shown in green and blue, respectively. Arrowheads indicate the presence of cytosolic dsDNA. Scale bars, 12.5 μm. c Percentage of cytoplasmic distribution of dsDNA 24 h after treatment of typhoid toxin in Henle-407 cells. Values are shown as mean ± s.d. (n = 3). One hundred cells were counted for each condition. Statistical analysis was performed using unpaired two-sided t-tests; ****P < 0.0001. d RT-qPCR analysis in THP-1-derived macrophages exposed to typhoid toxin. Statistical analysis was performed using unpaired two-sided t-tests (n = 3); ***P < 0.001, ****P < 0.0001. e IFN-β protein levels were assessed using ELISA in THP-1-derived macrophages after exposure to WT typhoid toxin and the mutants for 16 h. Data are presented as mean ± s.d (n = 3). Statistical analysis was performed using unpaired two-sided t-tests; **P < 0.01, ***P < 0.001. f RT-qPCR analysis in murine bone marrow-derived macrophages exposed to typhoid toxin. Statistical analysis was performed using unpaired two-sided t-tests (n = 3); *P < 0.05, **P < 0.01. g THP-1-derived macrophages exposed to typhoid toxin were analyzed using western blot analysis with antibodies against cGAS, phosphorylated STING, phosphorylated TBK1, phosphorylated p65, and β-actin as a loading control. h Analysis of the STING signaling pathway was performed using the STING-deficient THP-1-derived macrophages and its parental cells exposed to typhoid toxin. Cell lysates were analyzed by western blot with antibodies against cGAS, phosphorylated STING, phosphorylated TBK1, phosphorylated p65, p16INK4a, phosphorylated p53, and β-actin as a loading control. i, j The mRNA levels of proinflammatory components were assessed using RT-qPCR in the parental, STING knockout (KO) (i) or cGAS KO (j) macrophages exposed to typhoid toxin. Statistical analysis was performed using unpaired two-sided t-tests (n = 3); *P < 0.05, **P < 0.01 ***P < 0.001. The western blots shown in (g), (h) are representative of 3 independent experiments. Source data are provided as a Source data file.