FIG 2.

SCD1 activity is essential for HCV replication. (A) Huh7.5 cells were infected with Jc1 for 4 h and then incubated with medium containing either DMSO or various concentrations of SCD1 inhibitor. At 3 days postinfection, total cell lysates were immunoblotted with the indicated antibodies. (B) Subgenomic replicon cells derived from genotype 2a were treated with either DMSO or various concentrations of SCD1 inhibitor. Total cell lysates were immunoblotted with the indicated antibodies. Protein band intensities of HCV proteins/calnexin were analyzed by ImageJ. (C) Huh7.5 cells infected with Jc1 and subgenomic replicon cells derived from either genotype 1b or 2a were treated with either DMSO or various concentrations of SCD1 inhibitor. At 3 days after treatment, intracellular HCV RNA levels were quantified by quantitative PCR. Data represent average from at least three independent experiments. The asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001) from the value for the DMSO control. (D) Huh7.5 cells were treated with either DMSO or the indicated amounts of SCD1 inhibitor. Cell proliferations at the given time points were determined by MTT assay. Data represent averages from at least three independent experiments. (E) HCV subgenomic replicon cells (1b) were treated with the indicated concentrations of SCD1 inhibitor. At 3 days after treatment, total cell lysates were immunoblotted with the indicated antibodies. (F) HCV subgenomic replicon cells derived from genotype 1b were treated with various concentrations of SCD1 inhibitor for 72 h. Cell viability was assessed by MTT assay. Data represent averages from at least three independent experiments. ns, not significant compared to the value for the DMSO control. (G) Huh7.5 cells infected with Jc1 were treated with increasing amounts of SCD1 inhibitor. At 2 days postinfection, relative FFU from culture supernatants were determined compared to the value for the DMSO control. Data represent averages from at least three independent experiments. The asterisks indicate significant differences (**, P < 0.01; ***, P < 0.001) from the value for the DMSO control. (H) Huh7 cells harboring HCV subgenomic replicon were transfected with either vector or various constructs of SCD1. At 24 h after transfection, cells were further transfected with the indicated siRNAs. Total cell lysates harvested at 72 h after transfection were immunoblotted with the indicated antibodies (left panel). SCD1-SR, siRNA-resistant mutant SCD1; SCD1-SR-Hbox2A, siRNA-resistant mutant SCD1 with mutations in three histidine residues to alanine at histidine box 2. (Right panel) Quantification of NS3/GAPDH band intensity by ImageJ.