Estrogen Regulation of RNA and Protein

Culturing of cells was carried out as described previously46 (link)47 (link)48 (link)49 . For RNA or protein isolation, MCF7 cells in six-well tissue culture plates were maintained for 48 h in medium containing 10% charcoal dextran-stripped fetal bovine serum (CD-FBS). Cells were then treated without (Ethanol, EtOH, 0.01%) or with 10⁻⁹ M E2 and maintained for 3, 6 and 24 hours. At the termination, cells were subjected to total RNA isolation (Miniprep RNA isolation kit, ZymoResearch, Irvine, CA, USA) or protein extraction (NE-PER protein extraction kit, Thermo-Fisher). RNA and protein contents were assessed with NanoDrop (Thermo-Fisher) and Bradford Protein Assay (Bio-Rad Life Sciences Inc., Hercules, CA, USA), respectively.