Pgl3 basic

A luciferase reporter assay was performed as described previously [25 (link)]. Briefly, the luciferase reporter vector pGL3-basic (Addgene) was used to generate a luciferase reporter construct. Wildtype and mutant NKAP fragments containing the KpnI and XhoI restriction enzyme cutting sequences were synthesized. The synthesized DNA was digested with KpnI and XhoI, followed by insertion into the pGL3-basic vector. Next, hypoxic HUVECs were seeded at a density of 1×10⁶ cells/well in six-well plates. Then, hypoxic HUVECs were cotransfected with pGL3-NKAP-WT and miR-24-3p or pGL3-NKAP-MUT and miR-24-3p. The cells were maintained in an incubator for 24 h. Finally, relative luciferase activity was determined by a dual luciferase assay (Promega, U.S.A.).

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.