4.5. Reporter Gene Assay

ER-antagonistic activities of compounds were evaluated by a reporter assay using the Dual-Luciferase^® Reporter Assay system (Promega, Madison, WI, USA). MCF-7 cells were transfected with the firefly luciferase reporter plasmid containing three tandem copies of the estrogen-response element and control Renilla luciferase plasmid-SV40 using Lipofectamine LTX (Life Technologies, Inc., Gaithersburg, MD, USA). After 16 h, cells were treated with the indicated concentrations of test compounds in the presence of 17β-estradiol (3 nM). The firefly luciferase activity in cell lysates was determined based on the fluorescence intensity using a plate reader (ARVO SX1420, Perkin Elmer, Inc., Waltham, MA, USA) and normalized to the Renilla luciferase activity.