FIG. 1.

Decrease of HCV replication in Plk1 knocked down HCV replicon cell. (A) Schematic representation of the configuration of HCV-EV71I-Luc replicon construct. (B) Relative luciferase activity (L)/cell viability (M) ratio of Plk1 knockdown cells in the primary screen. The detailed experimental design is described in Materials and Methods. Means of two independent experiments including standard deviations of the means are shown. (C) Cellular RNA of HCVrep-His replicon cells was extracted at posttransduction day 3 and measured with quantitative RT-PCR. RNA levels of target genes were normalized by GAPDH RNA. Means and standard deviations of two independent experiments are shown. (D) Western blot analysis of HCV NS5A and Plk1 proteins in HCVrep-His replicon cells transduced by different shRNAs. Cell lysates were collected at day 4 posttransduction. The proteins were separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane, followed by standard Western blotting. Plk1 and HCV intensity levels are quantified by the densitometric scanning of the film using a bioluminescence imaging system (BioSpectrum Imaging System; UVP). The band intensity is normalized with the shLacZ control (set as 100%). (E) MTS assay of lentivirus-infected HCVrep-His replicon cells was performed on each day posttransduction until cell harvest (days 0 to 4) (mean ± standard deviation; n = 3). (F) The cell cycle of knocked down cells. The percentages of G₁/S/G₂ phases are indicated. shPlk1-2 and shPlk1-3 represent the lentiviruses harboring independent shRNAs targeted to different regions of Plk1 gene. shLacZ is the control virus targeting the LacZ gene.