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. 2010 Aug 30;207(9):1853–1862. doi: 10.1084/jem.20100239

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© 2010 Li et al.

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Figure 2. — PAD4 is required for chromatin decondensation and NET formation after LPS and H₂O₂ treatment. (A) Histone citrullination and nuclear morphology in untreated neutrophils. A small number (∼3.69%) of PAD4^+/+ neutrophils showed robust histone H3 citrullination (H3Cit) staining before stimulation. DNA dye Hoechst staining was pseudo-colored green. Decondensed chromatin was not observed before stimulation. Histone H3Cit or chromatin decondensation were not observed in PAD4^−/− neutrophils before stimulation. (B) LPS treatment induced histone citrullination and chromatin structural changes in PAD4^+/+ neutrophils. DNA was pseudo-colored green. Notice the swelling nucleus (top) and chromatin elongation (middle). In contrast, LPS treatment did not induce histone citrullination or chromatin structural changes in PAD4^−/− neutrophils (bottom). (C) Histone citrullination and neutrophil elastase staining colocalized with decondensed chromatin stained by DNA dye in PAD4^+/+ neutrophils after LPS treatment. (D) H₂O₂ treatment induced histone citrullination and chromatin structural changes in PAD4^+/+ neutrophils but not in PAD4^−/− neutrophils. For assays in A–D, peripheral blood neutrophils were purified from five PAD4^+/+ or five PAD4^−/− paired mouse siblings in each experiment, and at least three independent experiments for each treatment condition were performed.