Figure 5
From: Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2

Cooperation between the hypoxia-inducible factor (HIF)-2 and Ets-1 transcription factor. (A) HeLa cells were co-transfected with the −1091/+43 or −919/+43 pGL3-basic promoter construct (1 μg), pRL-TK plasmid (50 ng) and HIF-2α and/or Ets-1 expression vectors (1 μg each). Empty pcDNA3.1 was used to adjust total DNA content in all samples to 3 μg. The transfected cells were incubated in hypoxia and the promoter activity was assessed as described in Figure 2. Simultaneous expression of HIF-2α and Ets-1 showed synergic effect on endosialin promoter activity. (B) HeLa cells were transfected with the −235/+43 pGL3-basic promoter construct and its mutated variants with deletions of one (Δ−174/−147) or both (Δ−174/−91) EBS elements, respectively, and pRL-TK, HIF-2α and/or Ets-1 plasmids were co-transfected as above. The graph shows the promoter activity obtained in hypoxic cells only, as a similar profile was observed in the normoxic counterparts. (C) Chromatin immunoprecipitation assay was performed on FIB-3 cells under same conditions as described in Figure 4, except that primers flanking the ETS transcription factor-binding site (EBS) elements were used to detect binding of the HIF subunits. HIF-2α clearly bound to the EBS-containing region.