Fig. 3: Typhoid toxin activates the cGAS-STING signaling pathway by promoting mtDNA efflux into the cytosol. | Nature Communications

Fig. 3: Typhoid toxin activates the cGAS-STING signaling pathway by promoting mtDNA efflux into the cytosol.

From: Mitochondrial injury induced by a Salmonella genotoxin triggers the proinflammatory senescence-associated secretory phenotype

Fig. 3

a, Transmission electron microscopy of typhoid toxin-treated Henle-407 cells showing mitochondria (blue arrowhead). Scale bars, 500 nm. Cells are from 2 independent experiments. b, c Representative confocal images of TMRE staining in cells exposed to WT typhoid toxin and mutants. Scale bar, 15 μm (b). Mitochondrial morphology was classified into networked, intermediated, and fragmented categories using Imaris, with data representing the mean ± s.d (n = 3). At least 100 cells were counted for each condition (c). d, e Mitochondrial membrane potential measured by flow cytometry. THP-1-derived macrophages were treated with typhoid toxin. Sixteen hours after treatment, cells were stained with JC-1 dye (d). A histogram plot shows JC-1 intensity and quantification of JC-1 aggregate fluorescence indicating normal mitochondrial membrane potential. Data are presented as the mean ± s.d (n = 3) (e). Statistical analysis was performed using unpaired two-sided t-tests; ***P  <  0.001. f, g Fluorescent images of DNA (green), TFAM (red) and mitochondria (white) in Henle-407 cells exposed to typhoid toxin. Scale bar, 12.5 μm. Magnified images show the TFAM co-localizing with cytosolic DNA outside the mitochondrial network (f). Quantification of the ratio of cytosolic DNA foci distinct from those within the mitochondria relative to the total cytosolic DNA foci. Data are represented as the mean ± s.d. (n = 3), with 300 cells assessed for each condition (t-tests; *P < 0.05; **P  <  0.01) (g). h, i RT-qPCR of cytosolic mtDNA (cmtDNA) relative to total mtDNA in THP-1-derived macrophages (h) and Henle-407 cells (i) exposed to typhoid toxin using the mt16S primer set. Data are presented as the mean ± s.d. (n = 3) with statistical significance (t-tests; *P < 0.05, **P  <  0.01, ns, not significant). j RT-qPCR of total mtDNA in THP-1-derived macrophages exposed to typhoid toxin with or without ddC treatment. Mitochondrial DNA copy number was normalized by total nuclear DNA (ACTB). Data are presented as the mean ± s.d (n = 3) with statistical significance (t-tests; *P  <  0.05, **P  <  0.01). k RT-qPCR of indicated genes in THP-1-derived macrophages exposed to typhoid toxin in the presence of ddC treatment. Data are presented as the mean ± s.d (n = 3) with statistical significance (t-tests; *P  <  0.05, **P  <  0.01, ***P  <  0.001). Source data are provided as a Source data file.

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