Fig. 4.
Continuous GR activity is required to initiate circadian rhythm and maintain a synchronized phase. (A) Simultaneous treatment with RU486 antagonized dex-stimulated transcriptional oscillation. Cells were treated with 100 nM dex alone (red) or 100 nM dex and 1 μM RU486 simultaneously (gray) at time = 0 h (arrows). The samples were quantified as described previously (average and range of 2 independent experiments). (B) Addition of RU486 after dex treatment disrupts glucocorticoid-stimulated circadian rhythm. One micromolar RU486 (black) or vehicle (DMSO) (red) was added to the culture medium after 22 h of treatment with 100 nM dex (arrows). Transcript levels from 3 independent experiments were measured by quantitative PCR (mean ± SEM). (C) GR remains bound at GR-occupied Per2 GBS.6,7 during cycling of expression. ChIP experiments were performed in a time course after treatment with 1 μM dex. The data were processed as described previously and normalized to ChIP samples treated with vehicle (DMSO) for 8 h (mean ± SEM).