Fig 2: FMDV VP3 inhibits the activation of key molecules in the type-I IFN signaling cascade and suppresses antiviral gene expression. (A) Control plasmid or FMDV VP3 plasmid transiently transfected PK15 cells were infected with PR8-GFP (3MOI). At the indicated times after infection, TBK1, IRF3, STAT1, P65, and phosphorylated TBK1, IRF3, STAT1, and P65 protein levels in PK15 cell extracts were detected by immunoblotting, followed by the qRT-PCR for FMDV VP3, PR8-GFP virus NS1 protein, and ß-actin. The ß-actin was used to confirm the equal loading of proteins for immunoblotting. (B) HEK293T cells were transfected with control or FMDV VP3 plasmid together with GFP-IRF3 (green) plasmid, followed by infection with H1N1 influenza virus (1MOI). Cells were fixed at indicated time points and DAPI (blue) was used to stain the nuclei. Scale bar represents 5 µM. (C) The percentages of cells showing nuclear translocation of IRF3 were calculated by dividing the number of cells exhibiting nuclear expression of IRF3 by the total number of GFP-positive cells. (D) Control plasmid or FMDV VP3 plasmid transiently transfected PK15 cells were infected with PR8-GFP (3MOI). At indicated time points after infection, total RNAs were extracted from the cells, and expression of the indicated mRNA-encoding antiviral genes was analyzed by qRT-PCR. All the data are representative of at least two independent experiments, each with similar results, and the values are expressed as mean ± SD of two biological replicates. Student’s t-test; * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Fig 3: Analysis of the signaling pathway for cGAMP-mediated STING activation by DW2282 and KAS-08. (a) Western blot analysis for STING signaling pathway. Both DW2282 and KAS-08 were treated in THP-1 cells for 6 h in the absence or presence of cGAMP (1 µg/mL) stimulation. Black arrowheads indicate p-STING or p-STAT1 and gray double-arrowheads indicate non-specific band. (b) ISG reporter assay of DW2282 (up) and KAS-08 (down) in THP-1 WT, STING KO, and TBK1 KO cells. DW2282 and KAS-08 were treated in the range from 10 µM to 160 nM in the absence or presence of cGAMP (1 µg/mL). Luciferase signal was normalized by DMSO control. Graphs show the mean and SD. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 and ns: non-significant by Student’s t-test.

Fig 4: The expression of NLRC5, RIG-I and MDA5 but not that of NLRX1 is upregulated by CpG-A treatment in the human GEN2.2 pDC cell line. (A–E) GEN2.2 cells were treated with increasing concentration of CpG-A (0.25–1 µM) in a time dependent manner. The expression of NLRC5 and NLRX1 was measured at the mRNA level by Q-PCR (A) and at the protein level by western blotting (B,C). The changes in protein levels of RIG-I, MDA5, MAVS, and TBK1 were also analyzed after CpG-A treatments by western blotting (D,E). Representative blots are shown in (B,D). Data are shown as mean ± SD from 4 to 6 independent experiments in panels (A,C,E). Data were analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 1 µM CpG-A vs control; +p < 0.05, ++p < 0.01, +++p < 0.001, ++++p < 0.0001 0.5 µM CpG-A vs control; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 0.25 µM CpG-A vs control.

Fig 5: Mitotic SENP3 activates cGAS signaling.A Real-time PCR was used for the analysis of Ifnb1, Isg15, Il6, Ccl5, and Cxcl10 expression in SENP3-WT-MC38 cells (WT) or SENP3–9A-MC38(9A) cells. The data were normalized to Gapdh internal control and represented as mean with SD (n = 3 independent biological replicates). **p < 0.01, ***p < 0.001. B Cell lysates from SENP3-WT-MC38 cells (WT) or SENP3–9A-MC38(9A) cells were blotted with anti-SENP3, anti-phosph-TBK1(pTPK1), anti-TBK1, anti-phosph-P65(pP65), anti-P65, or anti-actin antibodies. C Real-time PCR was used for analysis of Ifnb1 and Isg15 expression in SENP3-WT-MC38 cells (WT) or SENP3–9A-MC38(9A) cells treated with or without RU.521 for 2 days. The data were normalized to Gapdh internal control and represented as mean with SD (n = 3 independent biological replicates). **p < 0.01. D cGAS in SENP3-WT-MC38 cells (WT) or SENP3–9A-MC38(9A) cells was knocked out by using Cas9/CRISPER system plasmid px459 encoding cGAS gRNAs (left panel). Real-time PCR was used for the analysis of Ifnb1 and Isg15 expression in these cells. The data were normalized to Gapdh internal control and represented as mean with SD (n = 3 independent biological replicates). **p < 0.01. E C57BL/6 mice were subcutaneously injected with cGAS-knockout SENP3-WT-MC38 cells (WT) or SENP3–9A-MC38(9A) cells (1 × 106/mouse). Tumor size was determined once in 3 days in two dimensions. Tumor growth curves represent the mean ± SD. Tumors were harvested and pictured on day 28 post-injection. Tumor weight was also measured immediately after harvest and the data from three independent experiments were shown in the bottom panel. Mean ± SD is indicated. **P < 0.01.