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. 2013 Sep 17;8(9):e75648. doi: 10.1371/journal.pone.0075648

Figure 4. Inhibition of HCV replication by ORP4 expression.

Figure 4

A. HCV subgenomic replicon RLuc cells were cultured in a 6-well plate and transfected with an indicated amount of pCMV-flag-ORP4 expression plasmid. pcDNA3, the cloning backbone vector, was used to equalize the total amount of plasmid DNA among the transfections; pCMV-βGal was included in each transfection to normalize the transfection variations. Cell lysates were prepared 48 hr post-transfection for the β-Gal activity and luciferase activity assay. The data are Means ± SD of triplicates and are representative of at least three independent experiments. B & C. Huh7.5 cells were cultured in a 24-well plate, first infected with HCV JFH-1 (0.5 MOI), then transfected with an increasing amount of pCMV-flag-ORP4 expression plasmid (62.5 ng, 250 ng, and 1 µg). An equal amount of pc3.GFP expression plasmid was included in all transfection to ensure a comparable transfection efficiency. Cells were harvested 3 days post infection for cell lysates and Western blotting using an anti-HCV core, Flag, or GFP antibody (B), or for MTT assay (C). 0* in panel C indicated that these cells were not transfected the ORP4 expression plasmid but infected with JFH-1. The data are Means ± SD of triplicates and/or are representative of at least three independent experiments.