Figure 3.

NETs formation in the lung during hepatic I/R is platelet-independent. (A) Representative immunofluorescence images by confocal microscopy showing NETs formation in the lung sections of mice after 1.5 hours of ischemia and 6 hours of reperfusion. Green: Ly6G; Red: Cit-H3; Gray: Actin; Blue: Nuclei. Arrow: released histone and DNA from the neutrophils. Scale bar, 100μm. (B) NETs formation in the lungs after 1.5 hours of ischemia followed by 6, 12, and 24 hours of reperfusion, as assessed by western blot analysis of Cit-H3 protein levels. (C) The correlation of MC38 cells arrested in the lung tissue at 6 hours post-hepatic I/R (solid red circles) or sham (hollow blue circles) with its serum level of MPO-DNA. (D) FACS quantitative data of MC38 cell numbers in lungs of mice at 12 hours post-injection in the lung of PAD4 deficient mice and in mice treated with DNase I. (E) The level of Cit-H3 protein was determined by western blot analysis in control or anti-CD41-treated mice after either sham laparotomy or 1.5 hours of ischemia and 6 hours of reperfusion. (F) Serum MPO-DNA complex levels were assessed in control or anti-CD41-treated mice in E. Data are presented as mean ± SEM from n = 3–10 mice per group. ns: not significant, ***P<0.001 and ****P < 0.0001.