FIG 1.

IFN-γ sensitivity of HCV replicons in Huh-7 and Huh6 cells. (A) Western blot analysis of stable HCV replicon cell lines derived from Huh6 and Huh-7 cells. Cells were treated with 10 ng/ml of IFN-γ or remained untreated for the indicated times. HCV NS3 protein was detected by using secondary antibodies labeled with IRDye and the LI-COR Odyssey system. Actin was used as a loading control. (B) Quantification of Western blot results in panel A. Band intensity was determined by using ImageJ software. Background was subtracted, and NS3 band intensity was normalized (norm.) to actin band intensity. Shown is the ratio of treated to untreated samples in percent. (C) Northern blot analysis of stable HCV replicon cell lines derived from Huh6 and Huh-7 cells. Cells were treated with 10 ng/ml of IFN-γ or remained untreated for the indicated times. RNA was used for Northern blot analysis with ³²P-labeled riboprobes specific for the neomycin resistance sequence, within the genomic HCV RNA or actin mRNA as a loading control. Two independent cell lines were analyzed for each genotype in the case of Huh6 cells and one was analyzed for Huh-7 cells. One representative blot is shown for each condition. (D) Quantification of Northern blot results in panel C. Band intensity was determined by using ImageJ software. Background was subtracted, and the HCV RNA band intensity was normalized to the actin band intensity. Two independent cell lines were analyzed for Huh6 cells and one was analyzed for Huh-7 cells. Shown is the ratio of treated to untreated samples in percent. (E) Long-term culture of stable Huh6 and Huh-7 replicon cell lines. Both replicon cell lines were seeded and then treated with G418 for selection of the HCV replicon. Additionally, 10 ng/ml of IFN-γ was added to select for IFN-γ-resistant cell populations. Cells were detached and counted, and the same amounts were reseeded every 3 to 4 days. Plotted is the ratio of cell numbers between G418- and IFN-γ-treated cells relative to that of cells treated with G418 alone. The experiment was performed twice with Huh6-derived cell lines and once with Huh-7-derived cell lines. Error bars indicate SDs. (F) Transient replication of HCV FLuc reporter replicons under treatment with IFN-γ. Naive Huh6 and Huh-7 cell lines were transfected with HCV FLuc reporter RNA (LucJFH1) and treated with IFN-γ at the given concentrations. Forty-eight hours after treatment, HCV replication was measured by luciferase assay. The experiment was performed 4 times; the results of one representative experiment are shown. Plotted are means between triplicate samples; error bars indicate SDs. rel, relative.