HIF-1alpha (HIF-1α) U2OS Redistribution Assay

• Designed to assay compounds for their ability to induce nuclear accumulation of HIF-1α
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

HIF-1 is a heterodimeric transcription factor consisting of HIF-1α and β subunits that permits activation of genes essential to cellular adaptation to low oxygen conditions (i.e. hypoxia). HIF-1β is constitutively expressed, whereas the expression of HIF-1α is maintained at low levels under normal oxic conditions (i.e. normoxia). During normoxia HIF-1α is hydroxylated on prolyl residues by the O₂ -dependent HIF1α prolyl-hydroxylase (PDH), which targets it for degradation mediated by the E3 ubiquitin ligase activity of the von-Hippel-Lindau (VHL) tumor suppressor protein. Under hypoxic conditions, O2 becomes rate limiting for prolyl hydroxylation, resulting in decreased ubiquitination of HIF-1α by VHL. This leads to accumulation of HIF-1α in the nucleus where it regulates a number of target genes involved in adaptation to hypoxic conditions. Since tumor cells are more hypoxic than normal cells and hypoxia is associated with poor prognosis and resistance to treatment, HIF-1α is considered to be a promising therapeutic target to kill tumor cells. Thus, manipulating the HIF-1 pathway has potential in treatment of several diseases including cancer and ischemia.

Figure 1. Accumulation of EGFP-HIF-1α. Cells were treated with 0.25% DMSO (control, left panel) or treated with 100 μM 2,2'-dipyridyl (right panel). Arrows indicate 2,2'-dipyridyl-induced accumulation of EGFP-HIF-1α detected by the image analysis algorithm.

Figure 2. Concentration response curves in the HIF-1alpha assay. Concentration response was measured in 9 point half log dilution series. Cells were incubated with test compound for 3 hours. Cells were then fixed and accumulation was measured using the Cellomics ArrayScan V^TI Reader and the RedistributionV3 BioApplication. % activity was calculated relative to the positive (100 μM 2,2'-dipyridyl) and negative control (0.25% DMSO). A) 2,2'-dipyridyl concentration response curve in the HIF-1α Redistribution assay (n=8). The EC₅₀ of 2,2'-dipyridyl is approximately 21 μM. B) Concentration response curves of reference compounds in the HIF-1α Redistribution assay (n=4). The EC₅₀ of CoCl2 is approximately 7 μM, the EC₅₀ of MG132 is approximately 5μM, and the EC₅₀ of DFOM is approximately 9 μM.