HIF-1α transcriptional activity

An HRE-luciferase construct (pGL4.42, 75 ng/well; Promega) expressing the luciferase reporter gene luc2p under the control of four HRE elements was co-transfected (FuGENE) into cells in a 96-well plate with a transfection control plasmid (pRL-CMV; 25 ng/well). Cells were exposed to glucose (24, 48 h, or chronic). A Dual-GLO Luciferase assay (Promega) was used to quantify luminescence 24 h post-transfection. Treatments to stabilize HIF-1α were performed 6 or 24 h before reading luminescence. Data from each well were normalized (pGL4.42/pRL-CMV relative luminescence units) and treated wells were compared with untreated wells (in triplicate).