FIG. 4.

Induction of cell cycle arrest by SIVmac239 Vpr and its mutant variants. HeLa cells were transfected with 15 μg of pCMV6Myc expression constructs and 10 μg of an expression plasmid for farnesylated GFP by using a calcium phosphate precipitate protocol. At 36 to 48 h after transfection, cells were harvested, fixed with 70% ethanol for at least 2 h on ice, and stained with propidium iodide (0.1% Triton X-100, 20 μg of propidium iodide/ml, 5 μg of RNase A/ml) for 30 min at 37°C. The DNA content of 30,000 cells was analyzed on a FACSCalibur (Becton Dickinson, Franklin Lakes, N.J.). Data acquisition and analysis were performed with CellQuest (Becton Dickinson) and ModFit (Verity Software House, Topsham, Maine) software with the doublet discrimination module activated. Samples were gated for GFP expression and exclusion of cellular debris. (A) Representative cell cycle profiles for the mutants in this series. The left peaks (M1) indicate cells in G₁, and the right peaks (M2) indicate cells in G₂/M; cells in S phase are shown between the G₁ and G₂ peaks. The percentages of cells in either G₁ or G₂/M were calculated by the ModFit program and are shown in the upper right of each graph. (B) Mean ratios of the numbers of cells in the G₂/M phase to those in G₁ (G₂/M:G₁ ratios) were calculated from three to six independent experiments, and standard deviations were deduced. For easy comparison, the ratio for wild-type Vpr was set as 1 and the relative values for Vpr mutants were deduced.