VCAM-1 expression in EC is regulated by HIF-2α. (A) The HIF-2α protein levels in kd/kd stromal cells (kd#3, kd#10) and MSS31 cells were examined by Western blot (left panel). Nuclear extracts were prepared from the cells cultured under normoxic (N, 20% O₂) or hypoxic (H, 1% O₂ for 6 hours) conditions. Right 2 lanes represent samples from kd#10 transfected with either HIF-2α cDNA (HIF-2α) or empty vector (vector). Quantification of the band intensities from 3 independent assays is shown in the middle column. The HIF-2α expression levels seen in MSS31 cells under normoxic conditions were normalized to a value of 1 as the standard. Right column represents quantitative RT-PCR analysis of VCAM-1mRNA. The VCAM-1 expression levels seen in MSS31 cells under normoxic conditions were normalized to a value of 1 as the standard (**P < .01, *P < .05). (B) The expression of VCAM-1 was examined by flow cytometry. Stromal cell lines derived from WT (wt#34 and wt#40) and kd/kd (kd#3 and kd#10) spleens were examined. Right 2 panels represent data from kd#10 cells transfected with either full-length VCAM-1 cDNA (VCAM-1) or vector alone (vector). (C) A coculture assay was performed using VCAM-1–transfected kd#10 cells. E13.5 fetal liver cells (2 × 10⁴ cells/well) were cultured on stromal cells in the presence of Epo (left) or Epo and SCF (right) for 4 days. The number of benzidine-positive colonies consisting of more than 200 cells was scored in each well and the average of colony number in triplicate wells for each stromal cell line is shown (**P < .01, compared with MSS31). (D) A luciferase reporter assay was performed using a WT VCAM-1-0.7 kb promoter (VCAM-1-Luc, top) and that with a mutation in HRE (VCAM-1-mut-Luc, bottom) as reporters. The pEF1–HIF-2α and pEF1-Arnt expression vectors were cotransfected with the reporter plasmid into 293T cells. Luciferase activities were measured for the VCAM-1-Luc (■) and VCAM-1-mut-Luc (□) reporters. The values shown are the averages of 3 independent experiments (means ± SD). The luciferase activity seen in 293T cells transfected with the reporter plasmid and empty vector was normalized to a value of 1 as the standard (*P < .05). (E) A chromatin immunoprecipitation assay was performed using anti-HIF-1α and anti–HIF-2α antibodies with WT and kd/kd EC under normoxic (N, 20% O₂) or hypoxic (H, 1% O₂ for 6 hours) conditions. Rabbit IgG was used as a negative control. Note that VCAM-1 expression was seen in WT EC using anti–HIF-2α antibody after treatment with 1% O₂. (F) Schematic models for roles of HIF-2α on erythropoiesis.