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. 2005 Feb 14;102(8):2986–2991. doi: 10.1073/pnas.0408707102

Fig. 3.

Fig. 3.

NS3/4A controls RIG-I signaling to NF-κB. (A and B) UNS3/4A cells, cultured to suppress (-NS3/4A) or induce (+NS3/4A) NS3/4A expression, were treated with IL-1, mock-infected or infected with SenV. (A) Cells were harvested at the times indicated, and extracts were subjected to immunoblot analysis to detect NS3, the phosphoserine 396 isoform of IRF-3 (IRF-3 Ser-396-P), total IRF-3, IκB-α, ISG56, and actin. (B) Cells were harvested 30 min post-IL-1 treatment or 16 h postinfection. Nuclear extracts were prepared and subjected to EMSA by using a DNA probe encoding the PRDII element of the IFN-β promoter to detect NF-κB DNA-binding activity. (C) Huh7 cells were cotransfected with plasmids encoding the PRDII-luciferase promoter construct and Renilla luciferase along with 50 ng of vector only or N-RIG expression plasmid and the indicated amount of NS3/4A expression plasmid. Cells were mock-infected or infected with SenV and processed for luciferase assay 16 h postinfection. Bars show the average relative luciferase and SD values from three experiments. (D) Huh7 2-3 cells harboring replicating genome-length RNA (HCV) or their IFN-cured Huh7 2-3c counterparts were cotransfected with plasmids encoding the PRDII-luciferase promoter construct and Renilla luciferase. Cells were mock-infected or infected with SenV and processed for luciferase assay. Bars show the average relative luciferase and SD values from three experiments. (E) Medium from cultures of mock or SenV-infected Huh7 2-3 (HCV) and Huh 2-3c cells (cured) was subjected to cytokine blot analysis by using the Cytokine Array III kit and the manufacturer's protocol (Ray Biotech, Norcross, GA). Numbers show the ratio of mock to SenV signal derived from averaged densitometric values of each spot. C denotes the reference control. (F) Huh7 2-3 (HCV) and Huh7 2-3c cells (cured) were mock-infected or infected with SenV for 16 h. Total RNA was extracted and subjected to microarray analysis by using Affymetrix U133A GeneChips. Bars show the quantified signal ratio of average hybridization levels for the indicated cytokine and chemokine mRNAs.