Fig. 4

HMGB1 from PMPs promote NETosis through autophagy. Control neutrophils were incubated with PMPs in the presence of with or without wortmannin (150 nM), 3-MA (5 mM), bafilomycin A1 (1µM), rapamycin (100 nM), Box A. The levels of H3Cit in the supernatant from each group was measured by ELISA (a) and neutrophils were stained with MPO (green) and LC3B (red) analyzed by confocal microscopy (b-g). Control neutrophils were incubated with PMPs in the presence of with or without Box A. h The expression of LC3B on treated neutrophils were detected by western blotting and neutrophils were stained with MPO (green) and LC3B (red) analyzed by confocal microscopy (i-n). o Control neutrophils were incubated with recombinant HMGB1 in the presence of with or without wortmannin (150 nM), 3-MA (5 mM), bafilomycin A1 (1µM), rapamycin (100 nM) and the expression of LC3B on treated neutrophils were detected by western blotting. The inset scale bars in b-g and i-n are 40 μm. Statistics, ordinary one-way ANOVA. Data are presented as the mean ± SD. ****P < 0.0001. PMN, polymorphonuclear