Fig. 5
Neutrophils are involved in the severe inflammation in the lung tissue of Stx12−/− mice. A KEGG pathway analysis of the differentially upregulated expressed genes in Stx12−/− mice (-log10 (padj) > 5.5) from the RNA-seq data. B The number of neutrophils in the blood from Stx12−/− and the littermate control (WT, Stx12+/+) mice. C The relative mRNA levels of neutrophil markers (Ly6g, MPO, ELANE, and CSF3R) and key chemokines for neutrophil recruitment and activation (CSF3, C3, CXCL1, CXCL2, CXCL5, and TNF) normalized to those of β-actin. WT n = 5, HO n = 5 independent samples. D Representative confocal images of immunofluorescence of frozen lung slice using antibodies against S100A9 and ly6g, the markers of neutrophils. Their increase of fluorescence intensity indicates the infiltration of neutrophils (WT n = 4; KO n = 3). E, F Quantification of the total fluorescence intensity of S100A9 and ly6g. It is relative to normalized WT intensity. G, H Quantification of fluorescence intensity of S100A9 and ly6g per cell. It is relative to normalized WT intensity. I Western blot analysis of the expression of IL-6, S100A9 and GAPDH in lung lysates from E19.5 Stx12−/− (HO) and the littermate control (WT, Stx12+/+) mice. J Relative quantification of IL-6 and S100A9 levels normalized to GAPDH. K IL-1β levels in the lung lysate from Stx12 − / − (n = 8) and the littermate control (WT) mice (n = 6) at E19.5 by enzyme-linked immunosorbent assay. L Relative mRNA levels of IL-6 from lung lysates of Stx12−/− (HO) and the littermate control (WT) mice. M Heatmap showing upregulated inflammation-related genes including neutrophil-related gene, interleukins, tumor necrosis factor (TNF)-related genes, complement(C)-related genes, colony stimulating factor (CSF)-related genes and chemokines in Stx12−/−(HO) and the littermate control (WT) mice lung tissues. The values are represented as log2 fold changes. The Stx12−/− mice and their littermate controls were obtained at embryonic day 19.5 (E19.5). The results are presented as the mean ± SEM; statistical significance was assessed by Student’s t- test