Effect of TSN on expression of HCV replicon. (A) HCV replicon cells were treated with various concentrations of TSN for 48 h. Replication levels of HCV RNA were analyzed by luciferase assay. Bars indicate luciferase activities relative to that of the drug-negative control. (B) Cell viability was determined by MTS assay. Bars indicate the value relative to that of the drug-negative control. (C) Western blotting analyses. The expression of NS5A and <t>beta-actin</t> was detected using anti-NS5A and anti-beta-actin antibodies. Densitometry of NS5A protein was performed, and the result is indicated as a percentage of the result for the drug-negative control. The assay was repeated three times, and a representative result is shown. (D) A bicistronic reporter gene plasmid, pCIneo-Rluc-IRES-Fluc, was transfected into Huh7 cells. The cells were cultured with TSN at the concentrations indicated, and dual luciferase activities were measured after 24 h of treatment. Values are displayed as ratios of Fluc to Rluc. In panels A, B, and D, the assays were done in triplicate and repeated three times. Error bars indicate means ± SDs.