Intermittent Hypoxia Affects the Spontaneous Differentiation In Vitro of Human Neutrophils into Long-Lived Giant Phagocytes

Figure 4

Effects of acute hypoxia on LC3B expression in giant phagocytes (Gϕ). Freshly isolated PMN were exposed for 24 h to intermittent hypoxia (IH, 56 cycles), sustained hypoxia (SH), or normoxia (N) and then cultured at normoxia for additional six days. (a) A representative western blot for LC3BI and LC3BII expression in freshly isolated PMN and normoxic-Gϕ. Densitometric analysis of LC3BII normalized over β-actin and normalizing by LC3BII/LC3BI ratio are presented. (b) Fixed cytospins were stained with LC3B (see Materials and Methods). Nuclei were stained with DAPI (blue). (c) Viable cells were labeled with MitoTracker Orange CMTMRos following fixation and LC3B staining. Mitophagy (LC3B positive structures containing mitochondria) appears as yellow spots (inserts in N and SH) with significant colocalization (MOC > 0.6) of LC3B/MitoTracker. Mitochondria without LC3B (red dots, arrows) were mainly noted in IH-Gϕ but also in SH-Gϕ. Representative data out of 4 independent experiments.

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