An estrogen-responsive element (ERE) reporter system consisting of three EREs in front of the Firefly luciferase gene (3X ERE TATA luc, a gift from Donald McDonnell, Addgene plasmid #11354 [16]) was used to assess ER transcriptional activity. The pRL Renilla luciferase control vector with CMV promoter (Promega) was used for normalization. Parental and fulvestrant-resistant cells were plated in 12-well plates, and the next day cells transfected with 600 ng 3X ERE TATA luc and 60 ng pRL Renilla using Lipofectamine 2000 (Invitrogen). The day after transfection, one well per condition was left untreated and one well was treated with 100 nM fulvestrant. After 24 h, Firefly and Renilla luciferase activities were assessed sequentially using the Dual-Luciferase Reporter Assay System (Promega) and a FLUOstar Omega reader.
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