Effects of IFNα and ATRA on the activity of a pIRE-containing promoter in the presence of Stat1α. COS-7 cells were transiently cotransfected with pIRE-TK-CAT (1 μg) and pnlsLACZ (0.5 μg) in the absence or presence of the indicated amounts of Stat 1α, RARα, or PML-RAR, as indicated. The total quantity of DNA transfected was always maintained at 10 μg by addition of an appropriate amount of the plasmid pBluescript. Sixteen hours after transfection, medium was changed and the incubation continued for further 36 hours with the indicated concentrations of IFNα or ATRA. At the end of each treatment, cells were obtained and processed for the measurement of CAT and β-galactosidase enzymatic activities. (A) Increasing amounts of pSG5-Stat1 construct were cotransfected with pIRE-TK-CAT and pnlsLACZ. Cells were incubated in the absence (□) or in the presence (▨) of an optimal concentration of IFNα (500 U/mL). The fold-increase relative to control conditions is indicated in parenthesis. (B) Increasing amounts of pSG5-Stat1 construct in the presence of a fixed amount of RARα (50 ng) were cotransfected with pIRE-TK-CAT and pnlsLACZ. Cells were incubated in the absence (□) or in the presence (▪) of an optimal concentration of ATRA (10−6 mol/L). The fold-increase relative to control conditions is indicated in parenthesis. (C) PSG5-Stat1 (0.5 μg), RARα (50 ng) were cotransfected with pIRE-TK-CAT and pnlsLACZ. Cells were incubated with increasing concentrations of ATRA, as indicated. (D) 50 ng each of RARα or PML-RAR were cotransfected with pIRE-TK CAT and pnlsLACZ in the absence or in the presence of PSG5-Stat1 (0.5 μg), as indicated. Cells were incubated with medium alone (□) or medium containing 10−6 mol/L ATRA (▪). All the results are the mean ± SD of three replicate dishes and are expressed as relative CAT activity, which is the ratio of CAT/β-galactosidase activity. All the data shown are representative of at least three independent experiments.
