Prl tk renilla luciferase plasmid

Cells were seeded in 24-well plate (2 × 10⁵ cells/well) 1 day before transfection and treatments were added accordingly to the experimental design (not treated, vehicle/DMSO 0.01% or fulvestrant 1 µM). For the putative promoter experiment, three different human genomic DNA regions representing putative ESR1-LBD promoter (pESR1-LBD-1/2/3) were cloned into pGL3.basic plasmid (Promega, USA; Supplementary Data 7). BC cells were co-transfected with pGL3.basic (NC) or p-ESR1-LBD reporter plasmids (750 ng) and pRL-TK Renilla Luciferase plasmid (75 ng) (Addgene, USA) by using Lipofectamine 2000 (Invitrogen, USA) and following reagent protocol. For ERα-driven transcriptional activity experiment, BC cells were co-transfected with 3x-ERE-TATA-Luciferase reporter plasmid (750 ng) and pRL-TK Renilla Luciferase plasmid (75 ng) (Addgene, USA) by using the same procedure described above. Cells were harvested 24 h after transfection and cell lysates were used for Dual-Luciferase® Reporter Assay System analysis, according to the manufacturer’s instructions (Promega, USA). Luciferase bioluminescence measurements were performed with the Veritas™ Microplate Luminometer (Promega, USA). For each sample, Firefly luciferase activity was normalized against Renilla luciferase activity.