FIGURE 5. The effect of cell density and 0.5% O₂ on activity of Gal4 DNA-binding fusion constructs.

Saos-2 cells were transiently co-transfected with the indicated Gal4 expression construct, pFR-Luc (a reporter vector in which the firefly luciferase gene is under the control of a minimal E1B promoter and upstream five copies of a Gal4 binding site, Stratagene), and pRL-CMV vector (internal control expressing Renilla luciferase, Promega) using Effectene reagent (QIAGEN). After exposure to the transfection mixture for 16 h, the cells were trypsinized, plated at the indicated cell density, allowed to adhere for 5 h, and exposed to normoxia or 0.5% O₂ for 24 h. Reporter assays were performed with the Dual-Luciferase Reporter Assay System (Promega). Activities of the Gal4-fusionconstructs are expressed as the average ratios of firefly to Renilla luciferase activities (± SD) from at least three independent experiments.