Fig 3.

The apoptosis rates in Jc1/Δ^XFP dual- and single-replicon-containing cells are similar. (A) Demonstration of the gating strategy used to analyze apoptotic cells. Huh7.5 cells were treated for 24 h with various concentrations of staurosporine to induce apoptosis or with DMSO vehicle (control). The cells were then stained with annexin-V APC and 7-AAD. Apoptotic cells are defined as 7-AAD⁻:annexinV⁺ cells. (B) No difference in apoptosis rates in dual- or single-replicon cells. Huh7.5 cells were transfected with Jc1/Δ^GFP and Jc1/Δ^EBFP2 RNA, followed 48 h later by FACS isolation of Jc1/Δ^GFP-only replicon-containing cells, Jc1/Δ^EBFP2-only replicon-containing cells, replicon-negative cells, and dual-replicon cells. Cells were stained with annexin-V APC and 7-AAD and analyzed at the given time points postisolation by flow cytometry. Cells were washed with medium 24 h postisolation and every 24 h thereafter, so the apoptosis rate reflects the preceding 24 h. Error bars indicate +SEM.