TRIM13 Positively Regulates the NF-κB Signaling Pathway Induced by Encephalomyocarditis Virus

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  Figure 1. TRIM13 promotes the proliferation of EMCV. (A,B) HeLa cells infected with EMCV for 0 h, 3 h, 6 h or 9 h were collected; 3D (A) and TRIM13 (B) expression was detected by RT-qPCR. (C,D) HeLa cells infected with EMCV for 0 h, 3 h, 6 h or 9 h were collected, and TRIM13 expression was detected by Western blot and densitometric analysis. (E,F) HeLa cells were transfected with 0.5 μg, 1.0 μg, or 1.5 μg of pcDNA3.1-3×Flag-TRIM13 plasmid and harvested 48 h post-transfection (hpt). TRIM13 expression was assessed by Western blot analysis (E), and cell viability was quantified by CCK-8 assay (F). (G–I) HeLa cells were transfected with 1.5 μg of pcDNA3.1-3×Flag-TRIM3 plasmid and then infected with EMCV (MOI of 1), transferred to 37 °C for 1 h, and then transferred to 37 °C for 9 h; cells and supernatant were harvested. TRIM13 expression was analyzed by Western blot analysis (G), and EMCV proliferation was assessed by RT-qPCR (H) and viral titration (I). (J,K) HeLa cells were transfected with TRIM13-siRNA-01, TRIM13-siRNA-02 or TRIM13-siRNA-03 and harvested 48 hpt. TRIM13 expression was detected by RT-qPCR (J) and Western blot analysis (K). (L) HeLa cells were transfected with TRIM13-siRNA-03…

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  Figure 2. TRIM13 selectively potentiates EMCV-induced pro-inflammatory cytokine expression in HeLa cells. (A–E) HeLa cells were transfected with 1.5 μg of pcDNA3.1-3×Flag-TRIM13 plasmid and then infected with EMCV (MOI of 1), transferred to 37 °C for 1 h, and then transferred to 37 °C for 9 h; cells and supernatant were harvested. TRIM13 (A), IL-6 (B,D) and TNF-α (C,E) expression were assessed by RT-qPCR (A–C) analysis and ELISA analysis (D,E). (F–J) HeLa cells were transfected with siRNA-03 specifically targeting TRIM13 and then infected with EMCV (MOI of 1), transferred to 37 °C for 1 h, and then transferred to 37 °C for 9 h; cells and supernatant were harvested. TRIM13 (F), IL-6 (G,I) and TNF-α (H,J) expression were assessed by RT-qPCR (F–H) analysis and ELISA analysis (I,J). (K,L) HeLa cells were transfected with 1.5 μg of pcDNA3.1-3×Flag-TRIM3 plasmid and then transfected with poly(I:C)-HMW (4 µg/mL), poly(I:C)-LMW (4 µg/mL), or LPS (1 µg/mL) and harvested 24 hpi. IL-6 and TNF-α expression were analyzed by RT-qPCR analysis (K) and ELISA analysis (L). (M–R) TRIM13-overexpressing (M–O) and TRIM13-knockdown HeLa cells (P–R) were infected with VSV (MOI of 1) and harvested 9 hpi. IL-6…

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  Figure 3. TRIM13 promotes EMCV proliferation by regulating the NF-κB pathway. (A,B) HeLa cells were transfected with pcDNA3.1-3×Flag-TRIM13 plasmid or TRIM13 siRNA-03 plasmid and then infected with EMCV (MOI of 1), transferred to 37 °C for 1 h, and then transferred to 37 °C for 9 h; cell pellets were harvested to detect the level of protein changes. (C,D) HeLa cells were transfected with pcDNA3.1-3×Flag-TRIM13 plasmid or TRIM13 siRNA-03 plasmid and then infected with VSV (MOI of 1), transferred to 37 °C for 1 h, and then transferred to 37 °C for 9 h; cell pellets were harvested to detect the level of protein changes. (E–I) HeLa cells were transfected with pcDNA3.1-3×Flag-TRIM13 plasmid and then infected with EMCV (MOI of 1), transferred to 37 °C for 1 h, and then transferred to 37 °C for 9 h; cell pellets were harvested. MDA5 (E), MAVS (F), TBK1 (G), IRF7 (H) and IκBα (I) expressions were assessed by RT-qPCR. (J–N) HeLa cells were transfected with TRIM13 siRNA-03 plasmid and then infected with EMCV (MOI of 1), transferred to 37 °C for 1 h, and then transferred to 37 °C for 9 h; cell pellets were harvested. MDA5 (J), MAVS (K), TBK1 (L), IRF7 (M) and IκBα (N) expressions were assessed by…

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  Figure 4. TRIM13 promotes EMCV proliferation by regulating the NF-κB pathway. (A–I) HeLa cells were co-transfected with empty pcDNA3.1 (vector) or pcDNA3.1-TRIM13 together with the indicated expression plasmids encoding Flag-MDA5, Flag-IRF7, Flag-MAVS, Flag-TBK1, Flag-IKKα, Flag-IKKβ, Flag-TRAF6, Flag-TRAF3 or Myc-IκBα for 48 h; cell pellets were harvested. Luciferase reporter assays were employed to evaluate the regulatory roles of MDA5 (A), MAVS (B), TBK1 (C), IRF7 (D), IKKα (E), IKKβ (F), TRAF3 (G), TRAF6 (H) or IκBα (I) on NF-κB promoters. (J–L) HeLa cells were co-transfected with empty pcDNA3.1 (vector) or pcDNA3.1-TRIM13 together with the indicated expression plasmids encoding Flag-MDA5, Flag-MAVS or Myc-IκBα for 48 h; cell pellets were harvested. Luciferase reporter assays were employed to evaluate the regulatory effects of MDA5 (J), MAVS (K) or IκBα (L) on IL-6 promoters. (M,N) HeLa cells were transfected with 0.5 μg, 1.0 μg, or 1.5 μg of pcDNA3.1-3×Flag-TRIM13 plasmid and harvested 48 hpt. TRIM13 (M) and IκBα (N) expression were assessed by RT-qPCR analysis. (O,P) HeLa cells were transfected with 0.5 μg, 1.0 μg, or 1.5 μg of pcDNA3.1-3×Flag-TRIM13 plasmid and harvested 48 hpt.…

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  Figure 5. TRIM13 promotes IκBα ubiquitination. (A) Co-localization of TRIM13 and IκBα proteins in HeLa cells and then infected with EMCV for 9 h. (B) Co-localization of TRIM13 and IκBα proteins in HeLa cells expressing TRIM13 and IκBα for 48 h. Nuclei were stained using DAPI, respectively. Data are representative of at least three independent experiments. (C) Co-IP and IB analysis of the interaction between TRIM13 and IκBα. (D) HeLa cells were transfected with pcDNA3.1-3×Flag-TRIM13 plasmid and subsequently infected with EMCV (MOI of 1). Nuclear–cytoplasmic fractionation was performed, and the subcellular distribution of VP1, TRIM13, IκBα, NF-κB, IRF7, Lamin B1 and GAPDH for cytoplasmic was analyzed by Western blot. Loading controls: Lamin B1 (nuclear fraction) and GAPDH (cytoplasmic fraction). (E) Immunoprecipitation analysis to investigate the regulatory effect of TRIM13 on the ubiquitination status of IκBα. (F) Immunoprecipitation assay to assess the influence of TRIM13 on K48-linked polyubiquitination of IκBα. (G) Immunoprecipitation experiment to evaluate the impact of TRIM13 on K63-linked polyubiquitination of IκBα.