Hypoxia inducible factor-1α (HIF-1α) in adenosine kinase (AK) repression. (A) Graphic representation of the putative AK promoter. Four putative hypoxia response elements (HREs) were identified (2 oriented forward [HRE], 2 on the antisense strand [rHRE], transcription start site [TSS]). The graph also displays the PCR product of the chromatin immunoprecipitation (ChIP) assays (AK-ChIP-1 or AK-ChIP-2). (B) ChIP assays were employed to examine HIF-1α binding to the human AK promoter in confluent HMEC-1 monolayers after hypoxia exposure. Controls for the ChIP assay included PCR performed with whole HMEC-1 cell genomic DNA (input), antibody control, samples precipitated with protein G sepharose beads alone (NX: normoxia, HX: hypoxia, 24 hours, 2% oxygen) and a known HIF-responsive gene (TLR2). (C) Confluent monolayers of control (HMEC-WT, ■) or oxygen-stable HIF-1α expressing (HMEC-ΔODD, ) HMEC cell lines or (D) HMECs with psiRNA repression of HIF-1α (HMEC-HIF1α, ) or control transfected cells (HMEC-Scr, ■) were exposed over indicated time periods to hypoxia (2% oxygen). AK or TLR2 transcript or protein levels were determined by real-time RT-PCR or Western blot analysis, respectively. Results are derived from 3 experiments (*P < .005 indicates differences between normoxia and hypoxia; #P < .001 between different cell types).