Effects of the HBZ-SP1 protein on HPV-18 transcription in HeLa cells. (A) The HBZ-SP1 protein inhibits HPV-18 transcription. HeLa cells (6 × 105) were cotransfected with 0.1 μg of LCR-Luc, 1 μg of pcDNA3.1(-)/Myc-His/lacZ (β-galactosidase-containing reference plasmid), 0.5 μg of pcDNA-JunB, and 0, 0.5, 1, or 2 μg of pcDNA-HBZ-SP1-Myc. The luciferase values are expressed as levels of fold activation relative to luciferase activity measured in cells transfected with pcDNA3.1(-)/Myc-His in the presence of the luciferase reporter gene without the early promoter P105. The total amount of DNA in each series of transfection was equal through the addition of pcDNA3.1(-)/Myc-His acting as filler DNA. Luciferase values were normalized for β-galactosidase activity. Values represent the mean ± S.D. (n = 3). (B) HBZ-SP1 protein expression in HeLa cells leads to the accumulation of cells in G1. At 24 h posttransfection, cells were harvested and GFP-positive cells (transfected with pEGFP-HBZ-SP1) were then analyzed for DNA content as described in Materials and Methods. The DNA content of EGFP-HBZ-SP1-transfected HeLa cells was then compared with that of untransfected cells by flow cytometry. The experiment shown here is representative of two independent experiments; the other experiment showed similar results. (C) The HBZ-SP1 protein blocks HeLa cell cycle progression through G1 phase. HeLa cells transfected or not with pEGFP-HBZ-SP1 were arrested in the cell cycle using a double thymidine block and restimulated with 10% FCS for the indicated times, harvested, and analyzed as described in panel B. The experiment shown here is representative of three independent experiments; the other two experiments showed similar results.