FIG. 1.

Selection and characterization of CsA-resistant HCV replicon cells in vitro. (A) Diagram of the selection scheme which combines antibiotic selection and live cell sorting. Note that the double selection with CsA and G418 was carried out for 3 weeks to obtain colonies, and the subsequent sorting of resistant cells was performed 3 days posttreatment. (B) CsA resistance profiles of the various replicon populations. GS5 cells, a population consisting of the top 15% of GFP-expressing GS4 cells sorted in the absence of CsA treatment, were used as the negative control. The CsA-resistant cells remained susceptible to IFN-α (C) and 2CMA (D). The GFP-based replicon cells were routinely treated with the indicated drugs for 4 days before being fixed for FACS analysis. The percentage of GFP-positive cells at the end of the treatments is normalized to the value of the untreated sample (set at 100%). (E) RS2 cells were resistant to long-term treatment with CsA. GS5 and RS2 cells were treated with 0.25 and 0.5 μg/ml CsA for 10 days, and cells were fixed for FACS analysis at days 3, 6, and 10 after the addition of the drug.