Estrogen Receptor Transactivation Assay

MCF7 cells were grown in 24 well plates in phenol red free DMEM supplemented with 10% (v/v) charcoal treated fetal bovine serum 48 h prior to estrogen (E2) treatment. Cells were transfected with pGL3-ERRβ, pEGFP-ERα [41 (link)], pEYFP C1-ERRβ [37 (link)], pRL-Renilla luciferase construct (Promega) in different combinations using jetPRIME-polyplus-transfection reagent (Polyplus transfection, New York, NY, USA) according to manufacture protocol. Post 24 h transfection cells were treated with 100 nM E2 and vehicle and were allowed to grow for 24 h. Luciferase assay was performed using Dual luciferase assay detection kit (Promega) according to manufacture protocol. Luciferase readings were obtained and were normalized with Renilla luciferase activity. The graph was plotted with normalized readings using GraphPad Prism software version 6.01.