Mode of siRNA introduction influences WNV RNAi susceptibility. A. Huh7.5-Rep cells. (Top) Diagram of the genetic structure of the pWN5'Pur replicon. (Bottom) Flow cytometric analysis of Huh7.5 cells that express the pWN5'Pur replicon. Only non-structural proteins (e.g., NS1 and NS3 but not E) are expressed. B. siRNA treatment of Huh7.5-Rep cells. Huh7.5-Rep cells were mock-transfected, transfected with TKO reagent complexed with 6337 or 6349 siRNAs, or electroporated with 6337 or 6349 siRNAs. Three days later, cells were processed for viral NS3 protein expression by flow cytometry using anti-NS3 antibody (right). Fold inhibition of NS3 antigen production was determined using the formula (% NS3 positive mock electroporated / % NS3 positive siRNA electroporated). C. RNA analysis of Huh7.5-Rep cells electroporated with siRNA. Huh7.5-Rep cells were electroporated with 6349 or 7353 siRNA as described in Materials and Methods. Three days later, total cellular RNA was collected and viral RNA was assessed. Fold inhibition was determined by dividing the amount of viral RNA in mock electroporated samples to the amount of viral RNA in siRNA electroporated samples. The results are an average of three independent experiments and error bars indicate standard error of the mean.