Effect of ATRA on FR-β promoter–luciferase reporter activity in transfected 293 cells.

(A) The 293 cells were cotransfected with the FR-β promoter linked to a luciferase reporter gene and an expression plasmid for RARα, RARβ, or RARγ, together with a β-galactosidase expression plasmid as described in “Materials and methods.” At 8 hours after transfection, ATRA (1 μmol/L) was added, and the cells were cultured for a further 40 hours. Cells were then harvested and lysed with luciferase reporter buffer, and the luciferase activity was determined as described in “Materials and methods.” The relative luciferase activity was normalized to β-galactosidase activity. (B) Representative clones A and B of recombinant 293 cells stably transfected with the FR-β promoter–luciferase reporter construct (described in “Materials and methods”) were treated with ATRA (1 μmol/L) for up to 5 days in 6-well plates. The cells were harvested and lysed on days 1, 3, and 5 of the treatment, and the luciferase activity in the lysate was determined as described in “Materials and methods.” The relative luciferase activity was normalized to 1 mg of total protein.