Transfection and luciferase reporter assay

The estrogen-responsive element (ERE)₃–Luc reporter construct (Hall & McDonnell, 1999) containing three copies of the vitellogenin ERE was purchased from Addgene (plasmid no. 11354). The plasmid was purified using a Plasmid midi kit (QIAGEN, USA). Cells were grown to 80%–90% confluence and transiently transfected with TransIT-LT1 Transfection Reagent (Mirus Bio, USA). Firefly luciferase activity was measured using a Luciferase Assay System (Promega, USA), and luminescence was measured using a LKB 1251 Luminometer. Luminescence was normalized to the total protein content measured using the Bradford assay.