Fig 2: FGFR3 knockdown activates the NF‐kB pathway. (A, B) DEGs after FGFR3 knockout by siRNA‐ FGFR3 were identified in the RT112 and UMUC‐14 bladder cancer cell lines. (C, E) KEGG and GO analysis of upregulated genes. (D, F) KEGG and GO analysis of downregulated genes. (G) Heat map of genes in NF‐kB signaling pathway. (H) KEGG pathway analysis indicated that the TLR3‐NF‐kB pathway was activated.

Fig 3: USP19 acts at the level of TRIF. a Overexpression of USP19 inhibits TRIF-mediated ISRE activation. HEK293 cells were transfected with ISRE reporter and the indicated adapter expression plasmids together with a control or USP19 expression plasmid for 24 h before luciferase assays were performed. Graphs show mean ± SD; n = 3 independent samples. **P < 0.01 (unpaired t-test); N.S., not significant. b USP19 interacts with TRIF in overexpression systems. HEK293 cells were transfected with HA-USP19 and the indicated adapter expression plasmids. Twenty hours after transfection, co-immunoprecipitation was performed with anti-Flag or control IgG. The immunoprecipitates and lysates were analyzed by immunoblotting with anti-HA or anti-Flag. c Domain mapping of the interaction between USP19 and TRIF. HEK293 cells were transfected with the indicated truncations before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. The schematic presentations of USP19 and TRIF truncations are shown at the top. d Effects of USP19 and its truncations on poly(I:C)- and LPS-induced IFN-β promoter activation. The 293-TLR3 or 293-TLR4 cells were transfected with the IFN-β promoter reporter and Flag-USP19 or its truncations for 24 h, and then left untreated, treated with poly(I:C) (50 μg/mL) or LPS (100 ng/ml) respectively for 8 h before luciferase assays. Graphs show mean ± SD; n = 3 independent samples. **P < 0.01, ***P < 0.001 (one-way ANOVA followed by Dunnett’s test); N.S., not significant. e Endogenous USP19 is associated with TRIF. The 293-TLR3 or 293-TLR4 cells were left untreated or treated with poly(I:C) (100 μg/ml) or LPS (100 ng/ml) respectively for the indicated times. Cell lysates were immunoprecipitated with anti-TRIF or control IgG. The immunoprecipitates and lysates were analyzed by immunoblotting with indicated antibodies. Data are representative of three experiments with similar results. Source data are provided as a Source Data file. Error bars represent standard deviation of the mean

Fig 4: Representative images of Western blot. Western blot was used to detect the expression of HLA, STAT1, TLR3, CXCL11, CXCL10, and C3 in cells from the MOCK group, H1N1 group, and H1N1 + oseltamivir group. β-Actin was conducted as internal control gene.

Fig 5: Effects of TLR3 knockdown on IL-6 production induced by stimulation with IFN-γ and poly(I:C). Cells were transfected with 50 pmol/well of human TLR3 gene-specific siRNA or negative control siRNA. After 2 d of culture, cell lysates were subjected to quantitative RT-PCR (a) (n = 3) and western blot analysis for TLR3 (b) (n = 2). (c) Cells were transfected with control or TLR3 siRNA. One day after transfection, cells were pretreated with IFN-γ (10 ng/mL) for 12 h, followed by stimulation with vehicle (none) or poly(I:C) (1 µg/mL) for 3 h. I + P indicates poly(I:C) stimulation in IFN-γ-treated cells. IL-6 mRNA expression was examined by quantitative RT-PCR. Data were normalized to GAPDH mRNA levels. Values are shown as mean ± SEM (n = 3). (d) Cells were stimulated as in (c) for 6 h. IL-6 levels in the reaction medium were measured by ELISA. Values are shown as mean ± SEM (n = 3). *P < 0.05 and **P < 0.01 versus the response to control.