Fig 4.

Inhibition of PLA2G4C does not impair HCV entry and translation but does inhibit HCV RNA replication. (A) Huh7.5.1 cells were transfected with 20 nM siRNA for 48 h or pretreated with MAFP at a concentration of 160 μM and then transduced with HCVpp. The luciferase activities were assessed 48 h later. (B) Huh7.5.1 cells were transfected with 20 nM siRNA and then were transfected with the pHCV-IRES plasmid 48 h later. The ratio of firefly luciferase (F-Luc) activities to Renilla luciferase (R-Luc) activities was determined 48 h later. Alternatively, Huh7.5.1 cells were transfected with pHCV-IRES and treated with MAFP at a concentration of 160 μM. The error bars indicate the standard deviations of triplicates. (C and D) Lunet-Con1 cells were transfected with siRNA for 48 h to establish the knockdown effect before being replated. (C) Total RNAs were extracted and submitted for quantitative RT-PCR determination of PLA2G4C mRNA and HCV RNA levels after 96 h. (D) Whole-cell lysates were collected for Western blot analysis of PLA2G4C, HCV NS3, and β-actin at 96 h. PLA2G4C was concentrated by IP using an anti-PLA2G4C antibody before the Western blot assay. (E and F) Con1 cells were incubated in the presence of MAFP at various concentrations. At the indicated time points after treatment, cells were collected and submitted to a quantitative RT-PCR determination of HCV RNA levels (E) and to Western blot analysis of HCV NS3 and β-actin after 96 h of treatment with MAFP (F). The bars indicate the standard deviations of triplicates. The significant differences of the different groups are shown as * (P < 0.05), ** (P < 0.01), and n.s. (no significant difference).