Fig. 6

Effect of HIV-1 Rev in mammalian cells. a Stable expression of HIV-1 rev gene in 293VE632 cells. The inducible pZH-1-rev plasmid was transfected into 293VE632 cells. The rev gene was expressed following addition of muristerone A and confirmed by Western blot analysis using a HIV-1 Rev monoclonal antibody. Lane “Off”, rev-repressing 293VE632 cells; lane “On”, rev-expressing 293VE632 cells. Western blot shows a 13 kDa protein band that was reacted to the anti-Rev antibody. β-actin was used as a loading control. b The 293VE632 cells containing the pZH-1-rev plasmid were grown under gene-repressing (rev-Off) and gene-inducing (rev-On) conditions for up to 7 days. The cells were collected every 24 h, and the cell numbers were counted with Tryptan blue staining (Sigma-Aldrich) [69]. The growth curves were generated with an average of three different experiments. c Rev does not affect mammalian cell cycle. The 293VE632 cells transfected with pZH-1 and pZH-1-rev plasmids were cultured without muristerone A (rev-Off) or with muristerone A (rev-On). DNA contents of the cells were determined by flow cytometric analyses after 5 days of culture. The upright numbers are the percentage of cells in the G1, S and G2 phases of the cell cycle. The 293VE632 cells containing pZH-1 plasmid was used as a control (upper row).