Fig 2: Relevance of antiviral signaling adapters and ISG response during DOX-induced DNA damage response.A–C Percentage of dead A549 cells with functional KO of the indicated genes relative to total cells counted over time (A, B) or 36 h (C) post DOX treatment. See Fig. S2 for DMSO controls. D A549 were stimulated with 1 µM DOX or 1 ng/ml dsRNA for 8 h. Phosphorylated IRF3 (S396) was determined by western blot. Two replicate lanes per condition: A549 wild-type cells (left lanes) and A549 cells with a KO of an irrelevant gene (IRF1, right lanes). Uncropped image of western blot shown in Fig. S9A. E A549 cells were stimulated with 1 µM DOX or 2 µg/ml poly(I:C) for 12 h. Cellular distribution of IRF3 eGFP (cyan) and histone H2B (magenta) was visualized by immunofluorescence microscopy. F A549 cells were stimulated with 1 µM DOX or 10 ng/ml dsRNA for 24 h. IFIT1 mRNA transcripts were determined by qRT-PCR. A–C, F Data shown represent the results of at least three biologically independent experiments.

Fig 3: Effects of Set-Met on the expression of MAVS protein and the phosphorylation of IRF-3 induced by PDCoV. LLC-PK cells were incubated with PDCoV for 1 h, and 2, 4, 8, and 16 µM of Se-Met was added. The expression of MAVS protein and the phosphorylation of IRF-3 were detected by western blot. Results were mean ± SD for three individual experiments.

Fig 4: (A-D)The effects of ASFV p17 on nuclear translocations of IRF3 and NF-?B p65. The 3D4/21 cells grown on the coverslips in 24-well plates (1×105 cells/well) were transfected with IRF3-GFP (0.5 µg) and pCAGGS-p17-2HA (0.5 µg) or control vector (0.5 µg) (A, B), p65-GFP (0.5 µg) and pCAGGS-p17-2HA (0.5 µg) or control vector (0.5 µg) (C, D) using Lipofectamine 2000. At 18 h post translation, the cells were stimulated with transfection of 2’3’-cGAMP (1µg/ml) for another 6 (h) The cells on coverslips were sequentially probed with rabbit anti-HA mAb, goat anti-rabbit IgG H&L Alexa Fluor 594, counter-stained with nucleus marker DAPI. (A) The effects of ASFV p17 on nuclear translocations of IRF3 was observed by fluorescence microscope with the enlarged images shown on the right. (B) Statistical analysis of the ratios of IRF3-GFP in nuclear fractions vs cytoplasmic fractions. (C) The effects of ASFV p17 on nuclear translocations of p65 was observed by fluorescence microscope with the enlarged images shown on the right. (D) Statistical analysis of the ratios of p65-GFP in nuclear fractions vs cytoplasmic fractions. (E, F) 3D4/21 cells in 24-well plates were transfected with the STING siRNA or Control siRNA (50 nM) by lipofectamine 2000 for 24 h, followed by co-transfected p17, ISRE-Fluc and Rluc for 12 h, and then stimulated by cGAMP for another 12 h. (E) Effects of the activation of ISRE promoter were detected by luciferase measurement. (F) Effects of the phosphorylation of TBK1 (p-TBK1) were detected by Western blot. Fold change values represent the mean ± S.D. *P < 0.05, **P < 0.01, ns not significant.

Fig 5: (A, B) The effects of p17 on the interactions and cellular co-localizations between STING and TBK1/IKK?. (A) 293T cells in 6-well plate were co-transfected with pcGAS-HA (0.5 µg) and pGFP-STING (0.5 µg) with or without pdsRed-p17 (0.5 µg and 1 µg) for 24 h (B) 293T cells were co-transfected with pGFP-STING (0.5 µg) and pMyc-IKK? (0.5 µg) with or without pdsRed-p17 (0.5 µg and 1 µg) for 24 (h) The cell lysates were subjected for immuno-precipitation and Western blotting using the indicated antibodies. (C, D) The effect of p17 on the interaction between endogenous STING and TBK1. (C) 3D4/21 cells were stimulated by transfection of poly I:C (1 µg/mL) or 2’3’-cGAMP (1 µg/mL), respectively, for 12h. The cell lysates were subjected for immune-precipitation with rabbit anti-STING pAb, and subsequent Western blotting using the rabbit anti-TBK1 mAb and anti-STING pAb. (D) The pCAGGS-p17-HA (2 µg) were transfected into 3D4/21 cells for 12h and then stimulated by transfection of 2’3’-cGAMP (1 µg/mL) for another 12h. The cell lysates were subjected for immune-precipitation and subsequent Western blotting. (E, F) The effect of p17 on the cellular co-localization between STING and TBK1/IKKe was analyzed by IFA. GFP and dsRed expression in cells was directly visualized by confocal microscopy. The endogenous TBK1 and Myc-IKK were stained by rabbit anti-TBK1 mAb and anti-Myc, respectively, followed by Alexa Fluor plus 647 anti-rabbit secondary antibody. (G, H) The effects of p17 on the activations of IFN promoter (G) and NF-?B promoter (H) triggered by transfected STING (20 ng), TBK1 (20 ng), IKK? (20 ng) and IRF3-5D (20 ng). The fold changes are relative to vector transfected controls. Values represent the mean ± S.D. *P < 0.05, **P < 0.01, ns not significant.