Fig 2: The expression profile of STING signaling pathway-associated molecules in human and mouse CRC tissues and TCGA database. RNA was extracted from cancer and matched peri-carcinomatous tissues of CRC patients, as well as tissues from AOM/DSS treated mice and reverse-transcribed into cDNA. Then the gene expression levels of STING signaling pathway-associated molecules were determined by quantitative PCR. The data from the same patient were connected by straight lines. The data of the expression of STING signaling pathway-associated molecules were retrieved from the TCGA microarray database in Oncomine® platform. Expression of STING (A, B, C), IFI16 (D, E, F), DAI (G,H, I) and DDX41 (J, K, L) in human CRC tissues, tissues from AOM/DSS treated mice, and TCGA database. Control: matched peri-carcinomatous tissues, n=44 for clinical samples, data represent two independent experiments; n=7 for PBS-treated mice and n=5 for AOM/DSS- treated mice; n=22 for health normal controls and n=215 for CRC group. *p < 0.05, **p < 0.01, ***p < 0.001; NS: Not significantly different.

Fig 3: The protein levels of DNA sensors in human CRC tissues. Protein was extracted from cancer and matched peri-carcinomatous tissues of CRC patients, and then the levels of DNA sensors including STING, IFI16, DAI, DDX41, DHX9, DHX36 and DDX60 were determined by Western-blot. (A) The representative Western-blot results. The triangles point to the specific bands of the molecules. Densitometric analysis of band intensity of STING (B), IFI16(C), DAI(D), DDX41(E), DHX9(F), DHX36(G) and DDX60(H) was shown. Control: matched peri-carcinomatous tissues, CRC: colorectal cancer tissues, n=12. *p < 0.05; **p < 0.01; NS: Not significantly different.

Fig 4: Correlation between STING signaling-associated molecules expression and cancer stages. The expression data of STING signaling pathway-associated molecules were retrieved from the TCGA microarray database in Oncomine® platform and were grouped by stages for further analysis. Correlation between STING (A), IFI16 (B), DAI(C), DDX41 (D), IRF3 (E), and cancer stages were shown. n=22 for health control, 44 for stage I, 78 for stage II, 52 for stage III, and 23 for stage IV. Data were expressed as mean ± SEM, Log2 median-centered ratio expression. *p < 0.05.

Fig 5: Proposed mechanism by which influenza virus regulates STING-dependent antiviral immunity. Ion channel activity of influenza virus M2 or EMCV 2B protein stimulates cytosolic mtDNA release in a MAVS-dependent manner. These cytosolic mtDNA could be packaged into distinct levels of higher-order structures depending on the ratio of TFAM to mtDNA. Cytosolic DNA sensors cGAS and DDX41 may recognize influenza virus-induced cytosolic mtDNA to stimulate STING-dependent IFN-β gene expression. The STING-dependent antiviral signaling was amplified in neighboring cells though gap junctions. The NS1 protein of influenza virus could associate with mtDNA to evade host DNA sensing pathways