FIG 5.

Treatment of Suit2 cells with thymidine impairs the ability of chemical compounds blocking the cell cycle in G₂/M to improve VSV-ΔM51 replication. (A) Experimental design scheme. Suit2 cells were incubated with either 2 mM thymidine or medium with 10% FBS for 24 h. VSV-ΔM51 (0.1 PFU/cell) was used to infect cells for 1 h. After virus incubation, cells were washed with PBS. Medium containing either the vehicle, 500 nM paclitaxel (PAC), or 500 nM colchicine (COL) (plus 2 mM thymidine) was added for 72 h. (B) Kinetics of GFP expression by VSV over the time after the different treatments described above for panel A. The data are from two independent experiments, each performed in quadruplicates, and data represent the means and SD of the means (*, P < 0.05; ****, P < 0.0001; ns, nonsignificant). The significance of the data was determined using two-way ANOVA with a Tukey posttest at a 95% confidence interval for comparison of VSV plus paclitaxel or VSV plus colchicine versus VSV alone. (C) Suit2 cells were incubated with either 2 mM thymidine or the vehicle for 24 h. Cells were then infected (or mock infected) with VSV-ΔM51 (0.1 PFU/cell) for 1 h. After virus incubation, cells were washed, and medium containing either the vehicle, 500 nM paclitaxel, or 500 nM paclitaxel and 2 mM thymidine was added for 24 h. Light and epifluorescence microscopy of Suit2 cells were imaged. (D) Cell cycle stages were analyzed for cells described above for panel C using flow cytometry with DAPI staining to determine nuclear DNA content, which was used to calculate the percentages of cells in different cell cycle phases. Single cells were gated via DAPI area and DAPI width signals and analyzed from a DAPI area histogram. The data are representative of results from two independent experiments. FACS, fluorescence-activated cell sorter.