Hypoxia Signaling Pathway Analysis

MDA-MB-231 TNBC cells were seeded into 24-well plates and transfected with 500 ng of ODD-Luciferase-pcDNA3 (Addgene Plasmid # 18965, a gift from William Kaelin) [32 (link)], or HRE-luciferase (Addgene Plasmid # 26731, a gift from Navdeep Chandel) [73 (link)] in conjunction with 500 ng Renilla pRL-SV40P construct (Addgene Plasmid #27163, a gift from Dr. Ron Prywes) [74 (link)] using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After 18 h, cells were treated with either DMSO (vehicle), cisplatin (5 µM), metformin (25 µM), gefitinib (5 µM) or different combinations for 24 h, after which cells were lysed and both Firefly and Renilla luciferase activities were quantified using a Dual-Luciferase^® Reporter Assay System (Promega, Madison, WI, USA) following the manufacturer’s instructions.