FIG. 1.

Cell cycle analysis of HeLa cells that expressed C81. HeLa cells were transfected with pME18Neo that encoded Flag-tagged wild-type Vpr or Flag-tagged C81 or with the control pME18Neo-Flag together with (A) or without (B and C) the GFP expression vector pEGFP-N1. (A) Analysis of DNA content. Thirty-six hours after transfection, cells were stained with PI and cells that were GFP positive were analyzed by flow cytometry as described in the text. Arrowheads indicate peaks of cells at the G₁ and G₂/M phases. The G₂/M:G₁ ratio is indicated in the upper right of each graph. (B) Two-color immunofluorescence of cells that progressed through the S phase. Thirty-six hours after transfection, cells were incubated with BrdU to monitor DNA synthesis, stained with anti-BrdU rat MAb followed by FITC-conjugated anti-rat IgG, and stained with Flag-specific MAb M2 followed by Cy3-conjugated anti-mouse IgG for detection of cells that expressed Vpr. (C) Two-color immunofluorescence of cells that progressed through the very late S-to-G₂ phase. Thirty-six hours after transfection, cells were reacted with anti-MCM rabbit serum followed by FITC-conjugated anti-rabbit IgG and with Flag-specific MAb M2 followed by Cy3-conjugated anti-mouse IgG for detection of cells that expressed Vpr. The stained cells were visualized by confocal laser scanning microscopy. Arrowheads indicate Vpr-positive cells.