Figure 7.

Expression of HIF-2α protein in normal bone marrow macrophages and U937 cell line. Normal bone marrow macrophages show immunoreactivity with mAb 190b. In serial paraffin sections of normal bone marrow trephine staining with CD68 (using mAb PGM1; A), and mAb 190b (B) colocalize in macrophages but not megakaryocytes. U937 cells were cultured with or without PMA (1.6 × 10⁻ 8 M) and incubated in normoxia (N) or 0.1% hypoxia (H) for 4 hours. Whole cell extracts (75 μg) were prepared, separated by SDS-PAGE, transferred onto PVDF membrane, and analyzed with mAb 190b (C). For comparison a hypoxic cell extract of HIF-2α transfected HT1080 cells (50 μg) prepared after culture for 4 hours in 0.1% hypoxia was run in parallel (lane C). The dominant band detected in each case comigrated at the mobility predicted for HIF-2α. In both differentiated and undifferentiated U937 cells the band detected showed hypoxic induction. However, after differentiation the normoxic levels seen were comparable with those seen in hypoxia in undifferentiated cells. U937 cells were cultured with PMA (1.6 × 10⁻ 8 M) to allow differentiation into macrophages, incubated in normoxia or placed in 0.1% hypoxia for 4 hours, and immunostained with mAbs to HIF-2α (190b) and CD11c (KB 90). HIF-2α expression was absent after normoxic culture (D) but detectable after hypoxic culture (E). CD11c expression, confirming macrophage differentiation, was unaffected by normoxic/hypoxic culture (not shown). Original magnifications, ×400 (A and B) and ×300 (D and E)