FIG. 6.

Transcriptional activation by the GR AF-1 is dispensable for cell cycle arrest in SAOS2 cells. (A) The GR 30iiB mutant induces complete cell cycle arrest in SAOS2 cells. SAOS2-30iiB stable transformants were generated as described in Materials and Methods. SAOS2-30iiB clones were seeded on day 0 into six-well plates (20,000 cells/well) and cultured in the absence or presence of 100 nM Dex. Total numbers of viable cells were determined on the indicated days by using the trypan blue exclusion method. (B) Induction of p21 and p27 by the GR 30iiB mutant. SAOS2-GR(+) (wt GR) and SAOS2-30iiB (30iiB) cells were cultured in the presence of 100 nM Dex or ethanol vehicle for 3 days, and whole-cell extracts were prepared and subjected to immunoblotting for GR, p27, p21, and ERK. Note the induction of p21 and p27 by both the wt GR and the 30iiB mutant. (C) Time course of p21 mRNA induction. SAOS2-GR(+) (wt GR) and SAOS2-30iiB (30iiB) cells were treated with 100 nM Dex for 0, 15, 30, 60, and 120 min, and total RNA was isolated and subjected to Northern blot analysis with a ³²P-labeled p21 cDNA probe (top panels). Equal loading in each lane is demonstrated by ethidium bromide staining of the 28S rRNA (bottom panels).