Figure 2.

Silencing NTCP Expression Inhibits HCV Infection in Human Hepatocytes

(A and B) NTCP silencing in hepatoma cell lines. Huh7.5.1-NTCP cells were transfected with siRNA control (siCtrl) or siRNA targeting NTCP (siNTCP). siRNA efficacy was assessed 72 hr after transfection by western blot and IF (scale bar, 10 μm) (A), and cell viability was assessed using PrestoBlue reagent (B). 3 days after NTCP silencing, Huh7.5.1-NTCP cells were infected with HCVcc (Luc-Jc1). Infection was assessed after 72 hr by measuring luciferase activity. Results are expressed as means ± SEM percentage cell viability compared to cells treated with siCtrl from three independent experiments (one performed in sextuplicate and two performed in octuplicate; n = 22) and means ± SEM percentage HCVcc infection from four independent experiments performed in quadruplicate (n = 16) (B).

(C–F) Effect of shNTCP silencing in hepatoma cells. Huh7.5.1-NTCP cells were transduced with lentiviruses expressing shRNAs targeting NTCP (shNTCP1, shNTCP2, or shNTCP3) or control shRNA (shCtrl). shRNA efficacy was assessed 72 hr after transduction by western blot (C) or by western blot and IF (scale bar, 10 μm) (E). Cell viability was assessed using PrestoBlue reagent (D). Results are expressed as means ± SEM percentage cell viability from three independent experiments performed in octuplicate (n = 24). 3 days after NTCP silencing, Huh7.5.1-NTCP cells were infected with HCVcc (Luc-Jc1) (D and F), HCVpp (genotype 1b) (F), or VSVpp (F). Infection was assessed after 72 hr by measuring luciferase activity. Results are expressed as means ± SEM percentage HCVcc infection from three independent experiments (two performed in quadruplicate and one in sextuplicate; n = 14) (D), or they are expressed as means ± SEM percentage pseudoparticle entry from three independent experiments performed in triplicate (n = 9) (F) and as means ± SEM percentage HCVcc infection from three independent experiments performed in quadruplicate (n = 12) (F).