Fig. 5

GSK-650394 regulates NETs formation in CRLM mice with IR background. Using GSK-650394 and then a 90-min warm ischemia model was constructed in mice, and the liver was removed after 6 h of reperfusion. A The expression of SGK1 was detected by western blot in ischemic liver or LO2. (n = 3 samples/group). B Before constructing CRLM-IR mouse model, GSK-650394 was injected intraperitoneally, and peripheral blood neutrophils were then isolated after 2 weeks of CRLM-IR surgery. The expression of SGK1 in circulating neutrophils of CRLM mice with IR background was detected by cytometry, and DMSO was injected intraperitoneally as control group (n = 3 samples/group). C The expression of MPO, NE, PAD4, and CitH3 was detected by western blot (n = 4 samples/group). D Quantification of MPO, NE, PAD4, CitH3 grayscale values (n = 4samples/group). E Immunofluorescence staining of CD36, MPO, Ly6G, CitH3 in livers of CRLM mouse undergoing IR (n = 4 samples/group). Scale bars, 50 μm. F Quantification of immunofluorescence (n = 4 samples/group). G The isolated neutrophils from circulation of wild type mice were pretreated with GSK-650394 or DMSO. In vitro, NETs were induced by PMA (200 nM), co-culture with MC38 cell line supernatant, and LPS (100 ng/ml). NETs were detected by scanning electron microscope (n = 4 samples/group). Scale bars, 10 μm. H Immunofluorescence staining of CitH3 (n = 4 samples/group), scale bars, 10 μm. I Correlation analysis of serum MPO-DNA (ng/ml) and relative mRNA expression in liver of patients with CRLM after surgery (n = 16 samples). All data represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001****p < 0.0001