![Figure 2. SDHBx neutrophil survival is independent of HIF-1α expression and linked to uncoupling of the mitochondrial electron transport chain. Human peripheral blood neutrophils from healthy controls (HC) and SDHB patients were studied in parallel. (A-B) HIF-1α protein expression. Freshly isolated neutrophils and neutrophils aged for 4 hours in normoxia (N4) or hypoxia (H4) were lysed and separated by SDS-PAGE, the membranes were probed for HIF-1α and p38 MAPK expression, and densitometry on hypoxic samples was performed. Representative blot shown (A), with mean densitometry ± SEM, n = 6 (B). (C-D) Glucose uptake in healthy controls (C) and SDHB patients (D). Neutrophils were preincubated in glucose-free phosphate-buffered saline in normoxia (N) or hypoxia (H) for 1 hour before stimulation with 100 nM N-formyl-methionyl-leucyl-phenylalanine (fMLP) in the presence of 200 μM 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose (2-NBDG) for 20 minutes. Uptake was determined by flow cytometry (FL1 geometric mean fluorescence). Data represent mean ± SEM, n = 5. (E) Glycolytic capacity in healthy controls (filled) and SDHB patients (open). Neutrophils were cultured with or without glucose, 2-deoxy-d-glucose (2DG; 10 mM), or LPS (1 mg/mL) for 2 hours before stimulation with 100 nM fMLP, and peak extracellular acidification rates were determined by Seahorse. Data represent mean ± SEM, n = 4. (F) Intracellular reactive oxygen species. 2′,7′-Dichlorofluorescein fluorescence was quantified after 45-minute neutrophil culture in the presence or absence of fMLP (100 nM), and fold change in patient neutrophil fluorescence was calculated relative to healthy controls. (G-H) Electron transport. Ratios of oxidized to reduced NADP (G) and NAD (H) were measured by fluorometric enzyme cycling assay in freshly isolated and aged neutrophils (6 hours). (I) Apoptosis. Neutrophils were cultured for 20 hours in the presence or absence of 3-nitropropionic acid (3-NP; 0-2 mM), and apoptosis was determined by morphological appearance; data represent mean ± SEM, n = 4. P values were determined by unpaired (B), paired (C, D, and I), or 1-sample (F) Student t tests, or by 2-way ANOVA (G-H). ECAR, extracellular acidification rate; OD, optical density.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/127/21/10.1182_blood-2016-02-696922/4/2641f2.jpeg?Expires=1724234200&Signature=S3~gDIHADUPPc8sXo6tzoGDmtabeJcpqQWFv4LK1QwfwwEwkRd3wIa1xw1np8iGVY~Lhz1dhnryWmp-jOJo0tV5bSqMquKUNai8XHN7kyZZq1Lmr-GOK9m-hEnRY2wFTy7ilxEnMijE-Tz~oFo9RkZWy5q5QhbptqumBoZiGuLfli~h-ej9cPDFPrgCdIJiIFanSjHJ5-rM9f6cAK3QA5pyOdzDX7lRKynUsqszrbl2EtvRAMpnvilhsYZ3KFfydj5d6NwmcQBo~yb0XVXvX8xbo2LpHZJ2tKfeBE2HGn6FgU6xmdX3T6xz3gMvws6VeBD4rBPlNn~d~9Djxs2onSg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
SDHBx neutrophil survival is independent of HIF-1α expression and linked to uncoupling of the mitochondrial electron transport chain. Human peripheral blood neutrophils from healthy controls (HC) and SDHB patients were studied in parallel. (A-B) HIF-1α protein expression. Freshly isolated neutrophils and neutrophils aged for 4 hours in normoxia (N4) or hypoxia (H4) were lysed and separated by SDS-PAGE, the membranes were probed for HIF-1α and p38 MAPK expression, and densitometry on hypoxic samples was performed. Representative blot shown (A), with mean densitometry ± SEM, n = 6 (B). (C-D) Glucose uptake in healthy controls (C) and SDHB patients (D). Neutrophils were preincubated in glucose-free phosphate-buffered saline in normoxia (N) or hypoxia (H) for 1 hour before stimulation with 100 nM N-formyl-methionyl-leucyl-phenylalanine (fMLP) in the presence of 200 μM 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose (2-NBDG) for 20 minutes. Uptake was determined by flow cytometry (FL1 geometric mean fluorescence). Data represent mean ± SEM, n = 5. (E) Glycolytic capacity in healthy controls (filled) and SDHB patients (open). Neutrophils were cultured with or without glucose, 2-deoxy-d-glucose (2DG; 10 mM), or LPS (1 mg/mL) for 2 hours before stimulation with 100 nM fMLP, and peak extracellular acidification rates were determined by Seahorse. Data represent mean ± SEM, n = 4. (F) Intracellular reactive oxygen species. 2′,7′-Dichlorofluorescein fluorescence was quantified after 45-minute neutrophil culture in the presence or absence of fMLP (100 nM), and fold change in patient neutrophil fluorescence was calculated relative to healthy controls. (G-H) Electron transport. Ratios of oxidized to reduced NADP (G) and NAD (H) were measured by fluorometric enzyme cycling assay in freshly isolated and aged neutrophils (6 hours). (I) Apoptosis. Neutrophils were cultured for 20 hours in the presence or absence of 3-nitropropionic acid (3-NP; 0-2 mM), and apoptosis was determined by morphological appearance; data represent mean ± SEM, n = 4. P values were determined by unpaired (B), paired (C, D, and I), or 1-sample (F) Student t tests, or by 2-way ANOVA (G-H). ECAR, extracellular acidification rate; OD, optical density.
