FIG 5.

Efficient HCV entry is dependent on NUMB and its phosphorylation. (A) Huh-7.5 cells were transfected with an siRNA targeting NUMB or an NT control. NUMB protein levels were measured by a quantitative Western analysis. A representative membrane and combined data from two independent measurements are shown. Numbers represent relative NUMB/actin protein ratios normalized to the level of the NT control. (B) Cellular viability was measured in the indicated cells at 48 h posttransfection by alamarBlue-based assays. Data are relative fluorescence values normalized to the value of the NT control. (C and D) These cells were infected with either HCVpp or HCVcc, and luciferase activity was measured at 72 and 9 h postinfection, respectively. Data are relative luciferase values normalized to the NT control signal measured at the respective time point. (E) Huh-7.5 cells were transfected with an empty plasmid or a plasmid encoding the WT or T102A NUMB mutant and infected with HCVpp or HCVcc 48 h posttransfection. (F) Cellular viability was measured at 48 h posttransfection by alamarBlue-based assays. Data are relative fluorescence values normalized to the level of the WT NUMB control. (G and H) HCVpp entry and HCVcc infection in these cells were measured by luciferase assays at 72 and 24 h postinfection, respectively. Data are relative luciferase values normalized to the WT NUMB control signal measured at the respective time point. Means and standard deviations (error bars) of results from three independent experiments are shown. RLU, relative light units. *, P < 0.05; **, P < 0.01; ***, P < 0.001.