Figure 4.

ARB does not induce IFN antiviral responses. A, ARB does not induce RIG-I dependent signaling. FL-Neo replicon and Huh7 cells were co-transfected with IFNB-luciferase and GFP or IFNB-luciferase and constitively active RIG-N expressing plasmids for 3 hours. Transfection mixtures were then removed and cells were incubated with medium containing the indicated concentrations of ARB. Luciferase activity was measured 24 hours later. B, ARB does not induce ISRE transcription. FL-Neo and Huh7 cells were transfected with ISRE-luciferase reporter plasmids for 3 hours. Transfection mixtures were then removed and cells were incubated with medium containing the indicated concentrations of ARB for 20 hours. Cells were then treated or not treated with 100 U/ml of IFN-α (Roferon) for 4 hours before luciferase activity was measured. C, ARB does not induce Stat1 phosphorylation on the conserved tyrosine amino acid at position 701. Huh7 or Huh7.5.1 cells were treated with the indicated amounts of ARB and whole cell protein extracts were harvested 20 and 60 minutes later. Cells were also treated with 500 units per milliliter of IFN-α, or as a negative control for possible solvent effects, an equivalent volume of ethanol (Ctrl). The position of Stat1-Y01 is indicated with arrows. D, ARB does not induce IFN stimulated gene expression. FL-Neo cells were treated with 6 μg/ml ARB for 1 to 4 days, or treated with 100 U/ml IFN-α for 24 hours. Whole cell protein extracts were probed for Stat1, Stat2, and GAPDH protein levels.