Fig. 1.

(a) Huh7.5 cells stably expressing an ISRE-luciferase reporter gene were transiently transfected with 25 nM siRNAs targeting USP18 as described in Materials and Methods. After 48 h, a dilution series of IFN-α2a was added to the cells, the cells were incubated for a further 17 to 24 h, and the percent induction of ISRE-luciferase reporter activity by IFN-α2a was measured relative to non-IFN-treated baseline ISRE-luciferase controls and plotted against IFN-α2a concentrations. Each condition was tested at n ≥ 2 replicates, and each experiment was independently repeated at least 3 times. Nonlinear regression analysis of the IFN-α2a dose-response curve was used to calculate each EC₅₀ by using GraphPad Prism software (version 5.01). A representative experiment is shown. Error bars represent the ranges of replicate data points. The inset table shows the degree of USP18 expression calculated by RT-PCR (corrected for actin) and the IFN-α2a EC₅₀ for ISRE induction. (b) Huh7 cells harboring the 9B subgenomic HCV replicon were transiently transfected with the same USP18 siRNA reagents described above and as described in Materials and Methods. After 48 h, a dilution series of IFN-α2a was added to the transfected cells and incubated for a further 17 to 24 h. Luciferase activity was measured by using an Analyst plate reader, and the percent inhibition of replicon activity relative to nontransfected cells treated with 10 IU/ml IFN-α2a was measured for each USP18 knockdown. Dose-response curves were generated by plotting the percent replicon inhibition for each USP18 knockdown against the IFN-α2a concentration. Nonlinear regression analysis was used to calculate EC₅₀s from each dose-response curve by using GraphPad Prism (version 5.01). Each condition was tested at n ≥ 2 replicates, and each experiment was independently repeated at least 3 times. A representative experiment is shown. Error bars represent the ranges of replicate data points. The inset table shows the degree of USP18 expression calculated by RT-PCR (corrected for actin) and the IFN antiviral (AV) EC₅₀. (c) The fold changes (FCs) in ISRE induction were calculated by dividing the EC₅₀ values for siCONT by the EC₅₀ values for siUSP18. Similarly, FCs in IFN-α2a AV potency were calculated by dividing the EC₅₀ values for siCONT values by the EC₅₀ values for siUSP18 from the replicon assay. The USP18 expression levels were measured by RT-PCR and calculated for each siRNA after correction for actin. Error bars represent standard deviations (SD) of pooled EC₅₀ differences generated by GraphPad Prism (version 5.01). NT, not transfected.