Fig. 2

NETs fueled HCC experimental metastasis in a LPS-induced NET model. a LPS mobilized neutrophils, and this was not disturbed by DNase 1 in C57BL/6 mice (n = 5 each). The liver and lung single-cell suspension was analyzed for neutrophils (Ly6G) infiltration by flow cytometry. Representative plot (upper panel) and quantification (lower panel) were shown. b Immunofluorescence detection of DNA, H3cit, and Ly6G revealed NET formation of infiltrated neutrophils in situ in the LPS-induced NET model. Representative images of NETs in the lung were shown. Scare bar: 50 μm. c NETs were released by neutrophils from LPS-treated mice, and this was efficiently disrupted by DNase 1 in vitro. Neutrophils were isolated from saline/LPS-treated mice and incubated with/without DNase 1 for 4 h. Neutrophils were then fixed and stained for SytoxGreen to observe NETs. Scare bar: 20 μm. d Serum MPO-DNA level was elevated by systemic LPS administration and significantly reduced by DNase 1 abrogation in vivo. e, f Representative images of the liver (e) or lung (f) experimental metastasis and quantification in LPS-induced NET model (n = 5 each). Hepa1-6 cells were intraspleen or intravenous adopted after LPS and/or DNase 1 administration in C57BL/6 mice. Scare bar: 200 μm. Saline served as a control. *P < 0.05; **P < 0.01; ***P < 0.001. Data were presented as means ± SEM