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.

缺氧诱导因子（Hypoxia Inducible Factor, HIF）转录因子对细胞适应低氧环境及机体发育至关重要，但其同时也参与缺血性疾病与癌症的发生发展。为筛选靶向HIF通路的新型遗传调控因子或可用于临床治疗的小分子化合物，基于细胞的报告系统是当前常用的研究手段。本研究报道了一种新型、高灵敏度且通用性优异的报告系统——NanoFIRE，即纳米荧光素酶与荧光整合报告元件（NanoLuciferase and Fluorescent Integrated Reporter Element）。该系统在缺氧反应元件（Hypoxic Response Element, HRE）的调控下，可作为细胞内HIF活性的稳健传感器，能够以高重现性、高灵敏度的方式有效响应低氧环境与HIF通路化学诱导剂，在不同细胞类型中均可实现20至150倍的信号诱导，且Z'因子大于0.5。我们证实，通过替换调控纳米荧光素酶表达的应答元件，NanoFIRE系统可实现功能适配，并可用于检测HIF抑制因子（Factor Inhibiting HIF, FIH）以及无关转录因子孕激素受体的转录活性。此外，通过慢病毒介导的稳定整合，NanoFIRE系统可在包括原代细胞在内的多种细胞类型中发挥作用，进一步凸显了其广泛的适用性。综上，本研究结果表明，NanoFIRE是一种可靠的报告系统，可用于研究细胞内HIF及其他转录因子介导的信号通路，同时可应用于高通量筛选，以鉴定新型小分子化合物与遗传调控因子。