TABLE 1.

Analysis of stage of the cell cycle of HeLa cells that expressed either C81 or wild-type Vpr

Protein	G2/M:G1 ratioa		% of cells stained with antibodies against cell cycle markersb
			BrdU (S-phase marker)		MCM (late S-to-G2-phase marker)
	Expt 1	Expt 2	Expt 1	Expt 2	Expt 1	Expt 2
C81	0.01	0.02	20.1	12.1	72.0	75.6
Vpr	1.02	0.75	19.0	16.8	18.3	32.4
Control	0.17	0.27	33.5	44.5	80.0	77.1

^aHeLa cells were transfected with pME18Neo that encoded Flag-tagged wild-type Vpr or Flag-tagged C81 or the control pME18Neo-Flag together with the GFP expression vector pEGFP-N1. Cells were harvested 36 h later and stained with PI. Cells were analyzed by flow cytometry. Data are presented after gating to eliminate cells in which GFP did not emit relative fluorescence. Percentages of cells in the G₂/M phase and G₁ phase were calculated. The G₂/M:G₁ ratios were calculated by dividing the percentage of cells at G₂/M by the percentage of the cells at G₁. 

^bHeLa cells were transfected with pME18Neo that encoded Flag-tagged wild-type Vpr or Flag-tagged C81 or the control pME18Neo-Flag. To detect cells that progressed through the S phase, 36 h after transfection, cells were incubated with BrdU, stained with anti-BrdU MAb followed by FITC-conjugated rat IgG and stained with an anti-Flag MAb followed by Cy3-conjugated mouse IgG. To detect cells that progressed through very late S-to-G₂ phase, 36 h after transfection, cells were treated with PBS that contained 0.05% Triton X-100, stained with anti-MCM rabbit serum followed by FITC-conjugated rabbit IgG, and stained with an anti-Flag MAb followed by Cy3-conjugated mouse IgG. Results are expressed as percentages of BrdU- or MCM-positive cells/Vpr or C81-positive cells.