Cyclosporin a

Huh-7 cells were electroporated with 5 µg of in vitro transcribed SGR RNA as done in the HCV replication assay. Electroporated cells were seeded in half of a 96-well plate per drug with a density of 3 × 10⁴ cells per well. At 5 h.p.e., standard cell culture medium was exchanged to DMEM complete containing 10% FCS, a constant end concentration of 0.01% DMSO and serial dilutions of telaprevir (S1538, Selleckchem), daclatasvir (S1482, Selleckchem), and cyclosporin A (C988900, Toronto Research Chemicals). In the case of the Intron A solution (Merck Sharp & Dome GmbH), serial dilutions in DMEM complete containing 10% FCS did not require the addition of DMSO as a solvent control. After 48 h of incubation, cell culture supernatant was removed and cells lysed on the plate by addition of 35 µL/well of luciferase lysis buffer (1% Triton X-100, 25 mM diglycine, 15 mM MgSO₄, 4 mM EGTA, and 1 mM DTT). Cell lysates were stored at −20°C until luciferase measurement was performed.