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. 2017 Jun 13;7:3409. doi: 10.1038/s41598-017-03257-z

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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JNK inhibition by SP600125 suppresses LPS-mediated NETosis. (A–C) NETosis kinetics was assessed by Sytox Green plate reader assay after activation with 25 nM PMA and 25 μg/ml LPS in the presence or absence of inhibitor. As shown in the %DNA release analysis, SP600125 (2.5, 5.0, 10 μM) suppresses LPS mediated NETosis in a dosage-dependent manner, while not in PMA mediated NETosis (n = 3–4; *p value < 0.05; Two-way ANOVA with Bonferroni’s post test conducted at each time point). Error bars in all the panels represent SEM. (D) Neutrophils were activated by PMA and LPS with and without SP600125 for 4 hours, immunostained, and imaged for myeloperoxidase (MPO) and DNA. MPO is visible around the nuclei in media control with or without SP600125. MPO co-localizes to NET DNA generated by LPS, PMA, and PMA with SP600125. Neutrophils treated with LPS and SP600125 do not show NETosis, and the nuclear morphology of these cells remains the same as that of the unstimulated control neutrophils (Blue, DAPI staining for DNA; Red, MPO; n = 3; scale bar 20 μm). See Supplementary Fig. S4 for low magnification images.