ERα target gene expression is cell cycle regulated in MCF-7 cells.

<p>A) FACS analysis after propidium iodide staining on asynchronous untreated cells (1), cells blocked in G1 by lovastatin treatment 20 µM for 32 h (2), in S phase by thymidine treatment 3 mM for 24 h (3) or in G2/M by nocodazol 50 ng/ml for 24 h (4). B) Real-time PCR analysis of <i>ESR1</i>, <i>PGR</i>, <i>CCND1</i> and <i>H2AFZ</i> gene expression in G1 (lovastatin), S (thymidine) and G2/M (nocodazol) cell cycle phases. 2×10⁶ of MCF-7 cells were seeded in 10 cm dishes and after 24 h submitted to specific cell cycle arrest drugs as in (A). Total RNA was extracted and reverse transcribed. The amount of analyzed genes cDNA was measured RT-qPCR divided by the amount of RLP0 cDNA. (n = 3) one representative experiment is shown. C) Real-time PCR analysis of <i>ESR1, PGR, CCND1</i> and <i>H2AFZ</i> gene expression. For lovastatin treatment cells were treated as in (B). For nocodazol/check-off treatment, 12×10⁶ cells were splited into two 140 cm dishes. After 24 h of nocodazol treatment (25 ng/ml), G2/M arrested cells are harvested by check-off.and seeded in a clean dish. After 7 hours in complete medium total RNA was extracted and reverse transcribed (n = 2).