Figure 1
RNF166 rather than RNF114, RNF125, or RNF138 potentiates RNA virus-induced IFN-β production.
(A) RNF166 positively regulates SeV- but not cGAS- and STING-induced activation of the IFN-β promoter. 293T cells (2 × 105) were transfected with IFN-β reporter (50 ng), pRL-TK Renilla luciferase plasmid (50 ng) and the expression plasmids indicated above. 12 h after transfection, cells were infected with SeV or left uninfected and luciferase assays were performed 24 h after infection. (B) RNF166 enhances the transcription of ISG15 and MX1. 293T cells (2 × 106) were transfected with empty vector or expression plasmid of RNF166 (2 μg), 24 h after transfection, the mRNA of ISG15 and MX1 were detected by Q-PCR. (C) HEK293T cell pools with stable knockdown RNF166 were generated by shRNA. Knockdown efficiency was determined by RT-PCR (left) and Q-PCR (right). (D) SeV-mediated activation of the IFN-β reporter was inhibited when the endogenous RNF166 was stably knocked down. Transfection and luciferase assays were performed as in (A). (E) SeV-induced IFN-β production by 293T-shRNF166-#4 and #5 cells was lower than by 293T-shGFP cells. Cells (1 × 106) were infected with SeV. 12 h after infection, cells were analyzed by Q-PCR (left) and the supernatants were collected for IFN-β bioassays (right). (F) HEK293T cell pools with stable knockdown of RNF114, RNF125 and RNF166 were generated using shRNA. Knockdown efficiency was determined by RT-PCR (left) and Q-PCR (right). (G) SeV-induced IFN-β production by cell pools with stable knockdown of RNF114, RNF125 and RNF138. Cells were infected and analyzed as in (E). (H) The knockdown efficiency of RNF166 in HeLa cells was determined by RT-PCR. (I–L) SeV-, EMCV-infection, poly (I:C)-transfection(4 ug) and Influenza A-infection, rather than dsVACV-transfection(4 ug) induced production of IFN-β by HeLa-shRNF166-#4 and #5 cells were lower than by HeLa-shGFP cells. Q-PCR and bioassays were performed at 12 h after treatment as in (E). Each graph represents the mean ± SD of three independent experiments done in triplicate. ***indicates P < 0.001; **indicates P < 0.01; ns (not significant) indicates P > 0.05.
