HIF-1 independence of hypoxic up-regulation of ALAS2.

(A) Firefly luciferase reporter gene constructs, containing either 3 copies of wild-type erythropoietin 3′ enhancer–derived HBSs or a wild-type ALAS2 promoter fragment (pTH10) were transiently transfected into HeLa cells with or without the addition of the iron chelators DFX and CPX. Cells were cultured for 16 hours under normoxic conditions. Results were normalized as described in Figure 1D. Mean ± SD of 3 independent transfections are shown. (B) Northern blot of DMSO-treated MEL cells cultered under normoxic conditions, with the addition of DFX, or under hypoxic conditions. The ribosomal protein L28 cDNA probe was used as a control for loading and transfer efficiency. (C) DNA-binding assays of nuclear extracts derived from HeLa cells cultured under normoxic or hypoxic conditions for 24 hours. HIF-1 DNA-binding activity was determined in nuclear extracts (5 μg) by means of oligonucleotide probes containing the predicted ALAS2 HBS or an erythropoietin 3′ enhancer–derived HBS. The specificity of HIF-1 DNA binding was confirmed by supershift analysis with the use of the anti–HIF-1α monoclonal antibody mgc3.25 wt indicates wild type; mt, mutant.