Cytokine priming does not protect cells from HCV NS3/4A-mediated inhibition of cytokine gene expression. (A) The effect of cytokine pre-treatment was studied in Luc-driven assay. The cells were left unprimed (non-treated) or primed with IFN-α, IFN-β, IFN-γ, (1000 IU/ml each) or TNF-α (20 ng/ml) for 18 h followed by transfection (6 h) with IFN-β promoter/Renilla luciferase reporter and HCV core or NS3/4A expression constructs. Transfected cells were infected with Sendai virus (MOI 5) for 16 h, cells were collected and luciferase activity was measured as indicated in the figure. The luciferase activity of the control sample was assigned to 1. (B) HEK293 cells were primed with IFN-α (1000 IU/ml) or left untreated for 16 h followed by transfection with Cardif and NS3/4A-wt or NS3/4A-S139A expression plasmids. Total cell lysate was prepared and Cardif and NS3/4A protein expression was analysed in cell lysates by immunoblotting. (C) RIG-I and Cardif mRNA was analysed in cytokine stimulated HEK293 cells. HEK293 cells were untreated (c) or stimulated with IFN-α, IFN-β, IFN-γ (1000 IU/ml each) or TNF-α (20 ng/ml) for 6 h or 16 h. Total cellular RNA was isolated and RNA samples (10 μg/lane) were analysed by Northern blotting with RIG-I and Cardif-specific cDNA probes. (D) HEK293 cells were untreated (c) or stimulated as above with IFN-α, IFN-β, IFN-γ or TNF-α for 24 h. Total cell lysate was prepared and RIG-I, Cardif, IKKε, IRF3 and IRF7 protein expression was detected by immunoblotting.