Hypoxia induces the production of mROS in neutrophils, which augments HIF-1α stabilization. (A-B) Neutrophils cultured in normoxia (21% O₂, red bars), 10% O₂ (light green bars), or hypoxia (1% O₂, blue bars) were stained with redox-sensitive dyes and fluorescence intensity analyzed using flow cytometry. (A) Neutrophil mROS levels were determined using MitoSOX Red after 1 hour in culture, n = 7. (B) Overall cellular ROS levels were quantified with DCF staining following treatment with fMLF, n = 8. (C,D) Untreated neutrophils (filled bars) and neutrophils treated with MitoTEMPO (open bars) were aged for 20 hours in normoxia or hypoxia and apoptosis determined through morphology, n = 7. Representative images at ×40 original magnification show hypoxia (top), hypoxia with MitoTEMPO (bottom). Red arrows indicate apoptotic cells. (E) Neutrophils were cultured for 4 hours in normoxia, 10% O₂, or hypoxia, sonication lysed, and proteins separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Membranes probed for HIF-1α relative to β-actin loading control, n = 4. (F) Neutrophils were cultured with or without rotenone (Rot, 2 μM), oxaloacetate (Oxa, 2 μM), antimycin A (AA, 10 ng/mL), DPI (10 μM), myeloperoxidase inhibitor-I (MPOi, 100 μM), or MitoTEMPO (MT, 100 μM) before lysis, SDS-PAGE, and membranes probed for HIF-1α relative to β-actin loading control, n = 6. Data represented as mean ± SEM. P values determined by (A-C) 2-way ANOVA or (E-F) 1-way ANOVA, *P < .05, **P < .01, *P < .001, ****P < .0001.