Cell Culture, Transfection, and Dual Luciferase Reporter Assay

Human embryonic kidney (HEK293) cells were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂. HEK293 cells were seeded at 10⁵ cells per well in a 24-well plate the day before transfection and co-transfected with 0.8 μg of the luciferase reporter construct and 0.1 μg of pRL-TK (Renilla luciferase) plasmid. Thirty hours after transfection, firefly and Renilla luciferase activities were measured consecutively using a dual Luciferase assay kit (Promega, Madison, WI). Renilla luciferase values were normalized to firefly and the ratio of Renilla/firefly values was reported. Each experiment was carried out more than three times in triplicate (repeatability = 0.93).