Figure 2.

Functional study of PHD2 mutants using luciferase reporter assays. (A) PHD2-dependent regulation of endogenous HIF in an assay based on a hypoxia response element reporter gene. Cells were co-transfected in a 12-well format with various amounts of pcDNA3-HA-PHD2 expression vectors (to enable the expression of the same amount of PHD2 proteins) in addition to pGL3 reporter vectors encoding firefly luciferase under the control of a sensitive hypoxia response element and Renilla luciferase as a control of transfection efficiency. Cells were placed in hypoxic conditions (1% O₂) for 4 h in order to accumulate endogenous HIFα before being collected. Results are given in percentage of firefly luciferase activity normalized to Renilla luciferase activity. The amount of HA-PHD2 transfected (PHD2) was quantified by immunoblotting using an anti-HA antibody. (B) The effect of PHD2 effect on HIF-2α protein stability in a one-hybrid reporter assay. Cells were cotransfected in a 6-well format with various amounts of pcDNA3-HA-PHD2 expression vectors (200, 100 and 50 ng), a Gal4-VP16-HIF-2αODD (amino acids 404-569) construct as well as a Gal4 response element-driven firefly luciferase reporter and a Renilla luciferase control plasmid. Twenty-four hours post-transfection cells were incubated for 16 h in normoxic or hypoxic conditions. Results are mean values of three independent experiments performed in triplicate.