Transient transfection and dual-luciferase assay

Luciferase assay for estrogen-responsive element (ERE) activity measurement was performed using a dual-luciferase reporter assay system (Promega, WI, USA) according to the manufacturer’s instructions. In brief, MCF-7 cells were seeded into 96-well plates at a density of 1 × 10⁴ cells/well. Cells were transfected with (ERE)₄-luciferase plasmid and SV40-Renilla luciferase plasmid using Lipofectamine 2000 reagent (Thermo Fisher Scientific). Transfection solutions were replaced after 24 h. Cells were then treated with and without 1 μM TAM and further incubated for 24 h prior to cell lysis and measurement of firefly- and Renilla-luciferase luminescence using a Stop-Glo reagent system (Promega; WI, USA). Each value was normalized to the Renilla luciferase control. Each data point generated represents the average of triplicate determinations.