Graph Pad prism files of all data published in Roennfeldt et al. (2023). Data points for each experiment represents the mean of an individual replicate (and thus each experiment was completed in triplicate with the mean +/- standard deviation graphed).

The Hypoxia Inducible Factor (HIF) transcription factors are imperative for cell adaption to low oxygen conditions and development, however, they also contribute to ischaemic disease and cancer. To identify novel genetic regulators which target the HIF pathway or small molecules for therapeutic use, cell-based reporter systems are commonly used. Here, we present a new, highly sensitive and versatile reporter system, NanoFIRE; a NanoLuciferase and Fluorescent Integrated Reporter Element. Under the control of a Hypoxic Response Element (HRE-NanoFIRE), this system is a robust sensor of HIF activity within cells and potently responds to both hypoxia and chemical inducers of the HIF pathway in a highly reproducible and sensitive manner, consistently achieving 20 to 150-fold induction across different cell types and a Z’ score > 0.5. We demonstrate that the NanoFIRE system is adaptable via substitution of the response element controlling NanoLuciferase and show that it can report on the activity of the transcriptional regulator Factor Inhibiting HIF, and an unrelated transcription factor, the Progesterone Receptor. Furthermore, the lentivirus-mediated stable integration of NanoFIRE highlights the versatility of this system across a wide range of cell types, including primary cells. Together, these finding demonstrate NanoFIRE is a robust reporter system for the investigation of HIF and other transcription factor mediated signalling pathways in cells, with applications in high throughput screening for the identification of novel small molecule and genetic regulators.