G2 arrest and cell death following adenoviral expression of Vpr.

<p>(A and B) HeLa cells were infected with adenoviral vector pAdeno-X/Flag-Vpr-IRES-ZsGreen1 or the control vector pAdeno-X/IRES-ZsGreen1 at MOI 100 and cultured for 72 h. (A) Cells were lysed and subjected to Western blot analysis with anti-Flag MAb, anti-caspase-3 polyclonal antibody, and anti-β-actin MAb. (B) Cells were fixed and stained in 3.6% formaldehyde containing Hoechst 33342. The DNA contents of ZsGreen1-positive cells were observed under a CELAVIEW microscope. Data points are means ± SD of triplicate samples. (C and D) Serum-starved HeLa/Fucci2 cells were infected with the adenoviral vector pAdeno-X/Flag-Vpr-IRES-ZsGreen1 at MOI 50 in DMEM containing 0.3% FCS. (C) At 23 h post-infection, cells were washed to remove uninfected virus and further cultured for 1 h in DMEM containing 10% FCS. Then, the cells were monitored by time-lapse imaging using the LCV110 Imaging System at 15 minute intervals at 73 h after release from serum starvation. Cells showing the cyan fluorescence of ZsGreen1 in entire cells established that the cells had been infected with adenoviral vector. Red or yellow fluorescence in nuclei show the cells are in G1 or S/G2/M phases of the cell cycle, respectively. High-magnification images of six cells (a to f) infected with the adenoviral vector are shown. The scale bar represents 5 µm. The percentages of each cell profile (a to f) of the ZsGreen1-expressing cells were estimated from triplicate dishes. At least 30 ZsGreen1-expressing cells were observed in every dish. Data are presented as means ± SD of triplicate dishes. (D) The dead cells were classified into G1, S and G2/M phases according to the color of the cell nucleus and cell morphology during observation. Data are presented as means ± SD of triplicate dishes. The asterisk indicates a statistically significant difference (*<i>p</i><0.05; **<i>p</i><0.01).