Fig. 4

The cGAS–STING pathway and Type I interferon pathway are activated in the lung tissue of Stx12^−/− mice. A Representative confocal images of immunofluorescence of frozen lung slices (30 μm) using antibodies against TOMM20 (mitochondria) and DNA. White arrowheads indicate cytosolic DNA foci. Scale bar, 10 μm. B Quantification of the average number of cytosolic DNA puncta per cell in the corresponding field. WT n = 3, KO n = 4 independent experiments. C The expression of cGAS, STING, the phosphorylation of TBK1 (p-TBK1), the phosphorylation of IRF3 (p-IRF3) and GAPDH by western blot in lung lysates from Stx12^−/− and the littermate control (WT, Stx12^+/+) mice. D Relative quantification of cGAS and STING levels normalized to β-tubulin. E Relative quantification of p-TBK1 and p-IRF3 levels normalized to β-tubulin. F Quantification of the levels of p-TBK1/TBK1 and p-IRF3/IRF3. G IFN-β levels of lung lysates from Stx12^−/− and the littermate control (WT, Stx12^+/+) mice (n = 5 per group) by enzyme-linked immunosorbent assay. H Heat map displays upregulated interferon stimulates genes (ISG) based on RNA-seq data of lung tissue. Rows represent individual ISGs and columns represent biological replicates. Color intensity reflects the level of gene expression and the values are represented as log2 fold changes. I Expression levels of ISGs by qRT-PCR from the lung tissues of Stx12^−/− (n = 6) and the littermate control (WT, Stx12^+/+) (n = 5) mice. The mRNA expression of target genes was normalized to that of β-actin. The Stx12^−/− mice and their littermate controls were obtained at embryonic day 19.5 (E19.5). The results are presented as the mean ± SEM; statistical significance was assessed by Student’s t- test