Transient transfections and quantitative RT-PCR analysis.

For the dominant negative assays, the U-2 OS cells stably expressing hGRα were maintained in Dulbecco's modified Eagle medium (DMEM)–F-12 (Invitrogen Life Technologies) supplemented with 5% heat-inactivated fetal calf serum, 50 units/ml penicillin, 0.05 mg/ml streptomycin, 2 mM l-glutamine, 0.2 mg/ml Geneticin (Invitrogen Life Technologies), and 0.2 mg/ml hygromycin B (Invitrogen Life Technologies). All cells were grown at 37°C and 5% CO₂ in a humidified incubator and passaged every 3 to 7 days as they approached confluence. The U-2 OS hGRα stably expressing cell line was plated in six-well plates at approximately 70% confluence 1 day before transfection. Cells were transfected with FuGENE 6 transfection reagent as described by the manufacturer (Promega, Madison, WI) using 6 μl of FuGENE 6 and 1.0 μg of DNA (pTRE2-hGRβ or pTRE2-GRβ′) per well or mock transfected (no DNA). After 18 to 24 h, the transfection medium was removed and replaced with fresh DMEM–F-12 maintenance medium containing charcoal-stripped fetal bovine serum instead of fetal calf serum, and the cells were then treated with 100 nM dexamethasone or vehicle (H₂O) for 6 h. Total RNA was isolated using a Qiagen RNeasy minikit. Real-time PCR was performed using a 7900HT sequence detection system with predesigned primer/probe sets available from Applied Biosystems (Foster City, CA), according to the manufacturer's instructions. The signal obtained from each gene primer/probe set was normalized to that of the unregulated housekeeping gene, cyclophilin B, primer/probe set (also available from Applied Biosystems). Each primer/probe set was analyzed with at least three different sets of RNA.

For the gene regulation assays, the U-2 OS cell lines stably expressing hGRβ and GRβ′ were plated in DMEM–F-12 charcoal-stripped fetal bovine serum and then treated for 6 h with 1 μM RU-486, and gene regulation was assayed as described above.