Fig 2: 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

Fig 3: 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 4: 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 5: Influenza virus stimulates DDX41-dependent IFN-β gene expression. a cGAS-293FT cells transfected with siRNA targeting DDX41 or control siRNA were infected with influenza virus for 24 h. Cell lysates were collected and blotted using the indicated antibodies (left panel). IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control (right panel). b, c cGAS-293FT cells were infected with WT (left panel) or ΔNS1 influenza virus (right panel) for 24 h in the presence or absence of LFM-A13 (100 μM) (b). WT or DDX41-deficient STING-A549 cells were infected with PR8 (left panel), or EMCV (right panel) for 24 h (c). IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control. d Pure cytosolic fraction prepared from digitonin extracts of mock- or ΔNS1 influenza virus-infected cGAS-293FT cells were treated with DNase I or RNase H. Cytosolic mtDNA was assessed by quantitative PCR. e HEK293FT cells were transfected with DNA extracted from DNase I- or RNase H-treated pure cytosolic fraction for 24 h. IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control. f STING-A549 cells transfected with siRNA targeting DDX41 or control siRNA were infected with PR8 virus for 24 h. Cell lysates were collected at 24 h post infection and blotted using the indicated antibodies (left panel). Pure cytosolic extracts were collected at 24 h post infection and analyzed for cGAMP by ELISA (right panel). g, h HEK293FT cells were transfected with siRNA targeting DDX41. Two days later, cells were transfected with the expression plasmid encoding Flag-tagged DDX41 or DDX41 (Y414F) mutant. Twenty-four hours after DNA transfection, the cells were infected with WT (g) ΔNS1 influenza virus (h) for 24 h. Cell lysates were collected at 24 h post infection and blotted using the indicated antibodies (left panel). IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control (right panel). These data are from three independent experiments (mean ± s.e.m.). **P < 0.01, ***P < 0.001; n.s., not significant (one-way ANOVA and Tukey’s test). Source data are provided as a Source Data file