Hypoxia-inducible factor 1-alpha (HIF1 alpha) is a constitutively expressed transcription factor that is degraded under normal oxygen tensions but stabilized when oxygen is limiting (hypoxia). Under hypoxic conditions, stabilized HIF1 alpha translocates to the nucleus and promotes the transcription of a host of genes that enable the cell to adapt to the lack of oxygen. Aspects of the HIF1 alpha mediated hypoxic response include promotion of angiogenesis and the switch from aerobic respiration to anaerobic glycolysis. Many of the HIF1 alpha responsive genes encode proteins that promote glycolysis and/or inhibit oxidative phosphorylation (known as the Warburg effect). An exciting and developing area of current cancer research is examining how HIF-mediated metabolic reprogramming promotes tumor growth and survival.

In most cases, HIF1 alpha will need to be stabilized to be measured (steady state levels of HIF1 alpha in non-hypoxic environments is exceeding low in most cell lines). This can be achieved by (a) creating a hypoxic environment (e.g. using a hypoxia chamber) or (b) by using chemical treatments that mimic hypoxia (e.g. cobalt chloride or deferoxamine). The sample data in this assay protocol was generated using deferoxamine (DFO). DFO is an iron chelator and disrupts the function the prolyl hydroxylases that degrade HIF1 alpha in normoxia. By disrupting the enzymes that degrade HIF1 alpha, DFO increases the abundance of HIF1 alpha protein.

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