Figure 1.

ROS and MPO Are Required for NE Translocation during NETosis

(A) Single confocal microscopy images of neutrophils from control, CGD, and ΔMPO donors. The neutrophils were stimulated for 60 min with C. albicans, immunolabeled for NE (red), and stained for DNA (Draq5, blue). Arrows indicate nuclear NE. Scale bars, 20 μm.

(B) NE release into the cytosol during NETosis measured by ELISA in cytosolic extracts derived from naive neutrophils alone (N) or PMA-activated neutrophils (NP) from control and ΔMPO donors. NE in the cytosol normalized to the total amount of NE in the cytoplasmic extract of each sample at time 0. Error bars indicate SD in triplicate samples; ^∗∗∗p < 0.001 between control and ΔMPO samples at 60 min. Cytoplasmic extracts were made by nitrogen cavitation, without detergent, to keep the granule membranes intact. Cytosolic extracts were made by ultracentrifugation of cytoplasmic extracts.

(C) Immunoblot of the degradation of exogenous histone H4 by cytoplasmic extract from naive neutrophils alone (N) or PMA-activated (NP) control and ΔMPO neutrophils. The cells were activated for the indicated time durations and H4 was incubated with the cytoplasmic extracts for 3 hr.

(D) Immunoblot against endogenous histone H4 in total cell lysates of naive neutrophils alone (N) from control or ΔMPO donors. Naive neutrophils (N) or stimulated with PMA (NP) or C. albicans (NC) for the indicated durations in the presence (+NEi) or absence of NEi. Full-length (H4, arrow) and proteolytically processed H4 (H4^∗, red arrow). C, C. albicans alone.

See also Figure S1.