FIG. 3.

TBC1D20 knockdown reduces HCV RNA levels in cells with established HCV replication without affecting cell viability. (A) A plasmid encoding a GFP-TBC1D20 fusion protein was transfected into Huh7 cells in the absence (left) or presence (middle and right) of siRNA duplexes (siRNA1 or siRNA3, respectively) designed against TBC1D20. (Top) Images were taken 24 h following transfection using a fluorescence microscope. (Bottom) Corresponding phase-contrast images. (B) Western blots of lysates from cells transfected as in panel A were probed with antibodies against GFP (top) or actin (bottom). (C) TBC1D20 knockdown using custom siRNAs. Cells with established replication of full-length HCV replicons (FLRP1) were transfected with a TBC1D20-targeting siRNA (siRNA3) or a control nontargeting siRNA. Total cellular RNA was harvested every 24 h and was used to determine TBC1D20 mRNA levels by quantitative real-time PCR. (D) RNA from the same experiment was used to determine HCV RNA levels by quantitative real-time PCR. The results were normalized to actin RNA levels. Values (means plus standard deviations) from three independent wells are shown and are expressed as percentages of the control. (E) Alamar blue assays for cell viability were performed at the indicated times following transfection of anti-TBC1D20 (siRNA3) or control (siCt) siRNAs.