The luciferase reporter plasmid, 3xERE-TATA-luc, was purchased from Addgene (Cambridge, MA, USA).27 (link) T47D-pMIG or T47D-ING4 cells were co-transfected with the linearized luciferase reporter plasmid and a neomycin resistance gene containing plasmid, pLNCx (Clontech, Mountain View, CA, USA) using Effectene (Qiagen Valencia, CA, USA) and were selected in the media containing 400 mg/mL Geneticin (Gibco, Billings, MT, USA). Cells plated at 50% confluency in a 24-well dish were hormone-deprived in the media containing 10% of charcoal-stripped FBS for 48 h and with or without 100 nM E2 for additional 24 h. Luciferase activity in 30 μL of cell lysates was measured using a Steady-Glo Luciferase Assay System (Promega Corporation, Fitchburg, WI, USA) and Victor3 luminometer (Perkin Elmer Life Sciences Products, Boston, MA, USA). Total protein concentration in cell lysates was measured using the BCA Protein Assay Kit (Pierce Thermo Fisher Scientific, Waltham, MA, USA). Luciferase activity was calculated as relative light units per microgram of protein and normalized to the luc2 gene copy number integrated into the genome as described previously.25 (link)