FIG. 5.

RU 486 functions as a GR agonist in the context of U2OS-GR(+) cells. (A) RU 486 inhibits proliferation of U2OS-GR(+) cells. U2OS-GR(+) cells were seeded on day 0 into six-well plates (15,000 cells/well) in duplicate and cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486. The total number of viable cells was determined on the indicated days. (B) Expression of Bcl2 is repressed in both Dex-treated and RU 486-treated U2OS-GR(+) cells. U2OS-GR(+) cells were cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 40 h, and expression of Bcl2 and ERK in whole-cell extracts was examined by immunoblotting as described in Materials and Methods. (C) ZK 299 is a pure GR antagonist in U2OS cells. U2OS-GR(+) cells were cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM ZK 299 for 40 h, and expression of Bcl2 and ERK in whole-cell extracts was examined by immunoblotting. (D) RU 486 is a weak agonist with respect to GR transcriptional activation in U2OS-GR(+) cells. U2OS-GR(+) cells were seeded in 6-cm-diameter dishes (120,000/dish) and transfected the following day via the calcium phosphate precipitation method with an XG₄₆TL reporter plasmid (4 μg/dish) and a pCMV-LacZ plasmid (0.75 μg/dish) as an internal control for transfection efficiency. Transfected cells were treated with an ethanol vehicle (−), 100 nM Dex, or 100 nM RU 486 (RU) for 12 h, and GR transcriptional activation was assessed via luciferase assay, normalized to β-Gal activity, and expressed as relative luminescence units (RLU). (E) RU 486 is a potent agonist with respect to GR-mediated transcriptional repression in U2OS-GR(+) cells. Cells were transfected with an XAP1TL reporter plasmid and a pCMV-LacZ plasmid, and GR transcriptional repression was assessed as described for panel D.