HRE-luc reporter assay

A promoter-reporter construct that contained 68 bp of a known hypoxia and HIF-1 regulated gene, enolase, containing a wild type hypoxia response element (HRE, 5’-RCTGT-3’), is a widely used approach for screening for HIF activators of different mechanism of action [29]. A cell-based assay with HRE-luciferase reporter system is based on transfected immortalized hippocampal cell line (HT22) and allows screening for a broad spectrum of compounds that include: activators of HIF transcription; activators of HIF binding to HRE; and effectors of HIF protein stability (PHD inhibitors, pVHL & proteasome inhibitors). The assay was performed the same way as for HIF ODD-luc reporter except the cells were plated at the density of 10,000 cell per well and incubated overnight.