Figure 2.

Longer infection duration increases superinfection of RVs. (A) RV supernatants collected at 24, 60 and 72 h were used to infect subconfluent A375 cells for the indicated duration (for example, 4–24 h). At the end of infection, medium was changed. The infected cells were selected with antibiotics at 24 h after infection. Cells were stained with crystal violet at 5 days after selection (a–c). Each assay condition was done in triplicate and representative results are shown. The stained cells were dissolved and quantitatively measured (a′–c′). ‘B+G’, blasticidin S resistance marker (BSD) and G418 double-selection. (B) Fluorescence detection of the superinfected cells. Subconfluent A375 cells were infected with the equal titers of RV-GN and RV-RB virus supernatant for 2, 4 and 8 h. Green fluorescent protein (GFP) and red fluorescent protein (RFP) expression was detected at 36 h after infection. The merged GFP and RFP expression is shown. Representative superinfected cells were indicated by arrows. (C) Superinfected cells maintain long-term co-expression of GFP and RFP markers. The RV-GN- and RV-RB-infected A375 cells were selected in BSD and G418 for 7 days and examined for GFP and RFP expression. Both signals were merged and representative superinfected cells are indicated by arrows. Representative images are shown.