Human neutrophils express HIF-2α, and expression is up-regulated by hypoxia, hydroxylase inhibition, and heat-killed bacteria. (A) Expression of HIF2A in freshly isolated neutrophils (t0) or cells cultured for 4 hours in normoxia (N) or hypoxia (H) was determined by PCR and agarose gel electrophoresis. A representative gel image of n = 3 is shown. (B) Fold change in expression of HIF2A and HIF1A following culture of human neutrophils in normoxia (filled bars) or hypoxia (open bars) for 6 or 12 hours. TaqMan analysis of cDNA was performed with data normalized to ACTB expression. Data show mean and SEM of fold change with respect to normoxic samples at 6 hours, n = 3, analyzed by ANOVA. (C-D) Expression of HIF-1α and HIF-2α is differentially regulated by hydroxylase inhibitors and up-regulated in response to heat-killed bacteria. Neutrophils were cultured in normoxia (Nx) with dimethyloxalylglycine (DMOG; 100 µM); deferoxamine (DFO; 300 µM); heat-killed Streptococcus pneumoniae (Spn; multiplicity of infection 10:1); LPS (100 ng/mL); or hypoxia (Hx) before being lysed. Proteins were separated using SDS-polyacrylamide gel electrophoresis (PAGE), and blots were probed for (C) HIF-1α and (D) HIF-2α. p38 MAPK was used as a loading control. (E-F) Densitometry analysis was performed on (E) HIF-1α and (F) HIF-2α blots using ImageJ software and normalized to p38 MAPK expression. Data are mean and SEM for minute n = 4.