Fig. 7

TBEV-induced RANTES expression is mediated by activation of IRF-3 pathway. a T98G cells were co-transfected with pGL2-220 and pRL-TK together with an empty vector or a dominant-negative mutant IRF-3 (IRF-3ΔN) or IRF-7 (IRF-7ΔN) and infected or uninfected with TBEV at an MOI of 1. Cells were harvested at 24 h post infection to measure luciferase activity. The results are expressed as fold induction of RANTES promoter activity relative to the basal level. Bars represent the means ± the standard deviations of three independent experiments. b The experiments were performed similar to those described in a except that a dominant-negative mutant IkBα plasmid (mIkBα) was used. Bars represent the means ± the standard deviations of three independent experiments. c Western blot analysis of phosphorylation state of IRF-3 in TBEV-infected cells. Whole-cell lysates were recovered from mock- and TBEV-infected T98G cells over a 48 h time course, and Western blot analysis was performed to examine levels of p-IRF-3, IRF-3, p-TBK1, TBK1, RIG-I, MDA5, IkBα, and β-actin. Three independent experiments were performed, and one representative result was shown. d Effect of RIG-I or MDA5 siRNA on RANTES expression in TBEV-infected cells. T98G cells were transfected with indicated siRNA. After 16-h incubation, the cells were mock-infected or TBEV-infected (MOI = 1) for 24 h. Supernatants were harvested and analyzed for RANTES protein expression (pg/ml) by ELISA (upper panel). Bars represent the means ± the standard deviations of three independent experiments. Expression of RIG-I or MDA5 proteins was assessed by Western blot analysis (lower panel). β-actin serves as a loading control. One representative result of three is shown. e T98G and CCF-STTG1 cells were infected with TBEV at an MOI of 1, followed by treatment with BX795, MG132, or DMSO vehicle in the absence of serum for 36 h. Levels of RANTES (pg/ml) released were determined by ELISA (upper panel). Bars represent the means ± the standard deviations of three independent experiments. Western blot was performed to examine levels of p-IRF-3 or IRF-3 (lower panel). One representative result of three is shown. *P < 0.05 indicates significant difference between groups tested. NS not significant