Time- and dose-dependent inhibition of viral replication by U18666A. (A) Huh9-13 cells were treated with 10 μg/ml of simvastatin or the indicated concentrations of U18666A for 72 h. w/o, without treatment. Viral replication was determined by RT-PCR. The graph shows the relative data from three independent experiments, with SEMs. (B) Huh7.5 cells were transfected with the J6-Luc construct and treated with 1,000 U of interferon alpha, 10 μg/ml of simvastatin, or the indicated concentrations of U18666A for 72 h. Viral replication was determined by luciferase reporter gene assay. The graph shows the relative data from three independent experiments, with SEMs. (C) Huh7.5 cells were transfected with the J6-Luc construct and treated with 2 μg/ml of U18666A for 72 h. Viral replication was determined by luciferase reporter gene assay at different time points. The graph shows the relative data from three independent experiments, with SEMs. (D) Huh9-13 and Huh7.5 J6 cells were treated with 2 μg/ml of U18666A or 1,000 U of interferon alpha for 16 h. Viral replication was determined by RT-PCR. The graph shows the relative data from at least three independent experiments, with SEMs. ns, not significant. (E) Western blot analysis of cellular lysates derived from Huh9-13 and Huh7.5 J6 cells which were treated with 2 μg/ml of U18666A or 1,000 U of interferon alpha for 16 h. For detection, specific antibodies against NS3, NS5A, and core were used. Detection of β-actin served as a loading control. (F) Densitometric quantification of Western blots shown in panel E. The graphs show the quantification of at least three independent experiments, with SEMs.
{}