American Society of Hematology

Figure 5.

$Figure 5. Oxidants produced during efferocytosis and efferosome acidification regulate efferosome maturation and proteolysis. (A) WT (black bars) and CGD (white bars) PEMs were preincubated (30 minutes) either with 100 μM TBHP (TB) or 1 mM Tiron. PEMs were then fed DQ-BSA–coated hANs for 15 minutes and the percentage of efferosomes showing DQ-BSA cleavage determined. PEMs were also pretreated with bafilomycin A1 for 30 minutes before feeding with hANs or DQ-BSA–coated hANs for 10 minutes and the fraction of efferosomes exhibiting (B) DQ-BSA-cleavage, (C) LC3 localization, and (D) Lamp1 recruitment was determined. Results from 4 independent experiments are shown as means ± SD. *P < .05; **P < .01, ***P < .001 by the Student t test. (E) Polystyrene beads coated with IgG and DQ-BSA were fed to PEMs at a ratio of 20:1 for 10 minutes. Some PEMs pretreated with 1 μM bafilomycin for 30 minutes before and during the addition of beads, as indicated. After imaging with confocal microscopy, the percentage of efferosomes containing cleaved DQ-BSA was calculated. Results from 4 experiments were shown. *P < .05 by Student t test.$

Oxidants produced during efferocytosis and efferosome acidification regulate efferosome maturation and proteolysis. (A) WT (black bars) and CGD (white bars) PEMs were preincubated (30 minutes) either with 100 μM TBHP (TB) or 1 mM Tiron. PEMs were then fed DQ-BSA–coated hANs for 15 minutes and the percentage of efferosomes showing DQ-BSA cleavage determined. PEMs were also pretreated with bafilomycin A1 for 30 minutes before feeding with hANs or DQ-BSA–coated hANs for 10 minutes and the fraction of efferosomes exhibiting (B) DQ-BSA-cleavage, (C) LC3 localization, and (D) Lamp1 recruitment was determined. Results from 4 independent experiments are shown as means ± SD. *P < .05; **P < .01, ***P < .001 by the Student t test. (E) Polystyrene beads coated with IgG and DQ-BSA were fed to PEMs at a ratio of 20:1 for 10 minutes. Some PEMs pretreated with 1 μM bafilomycin for 30 minutes before and during the addition of beads, as indicated. After imaging with confocal microscopy, the percentage of efferosomes containing cleaved DQ-BSA was calculated. Results from 4 experiments were shown. *P < .05 by Student t test.

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