Luciferase Reporter Assay

HeLa and HeLa-MX cells were seeded into a 6-well plate at 4 × 10⁵ cells per well and transfected with 2 μg of the luciferase vector (HRE-luc) containing hypoxia-responsive elements the next day, using the Turbofect Transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells were co-transfected with 100 ng of pRL-TK Renilla vector (Promega) to normalize for the transfection efficiency. Luciferase reporter construct containing three hypoxia response elements (HRE; 24-mers) from the Pgk-1 gene was a gift from Navdeep Chandel (Emerling et al., 2008) (Addgene plasmid #26731⁴; RRID:Addgene_26731). Reporter gene expression was assessed 48 h after transfection using the Dual Luciferase Reporter Assay System (Promega) and the Synergy HT reader with Gen5 software (BioTec Instruments). Luciferase activity was normalized against Renilla activity.