Neutrophil Elastase Antibody - #AF0010

Fig. 5. Effects of CS ion solution on modulation of the formation of NETs when FOXO3 was inhibited. (A) Western blot analysis demonstrating the activation of FOXO3 by CS in NETs. (B to D) Quantitative analysis of p-FOXO3 and t-FOXO3 protein expression levels as well as p-FOXO3/t-FOXO3 ratio based on Western blot. (E) The NET markers, including MPO-DNA, NE-DNA, cfDNA, and IL-1β, were measured in the medium after using CS ion solution and siFOXO3 by ELISA. (F) NETs treated with CS ion solution and siFOXO3 were stained with MPO (red), NE (green), and DAPI for visualization. Scale bars, 20 μm. n = 6, ###P < 0.001 versus control; *P < 0.05, **P < 0.01, ***P < 0.001 versus NETs; &P < 0.05, &&P < 0.01 versus NETs + CS + siFOXO3 (control: neutrophils not initiating NET differentiation; PMA: neutrophils initiating NET differentiation; PMA + CS: neutrophils initiating NET differentiation cultured with CS solution; PMA + siFOXO3: neutrophils initiating NET differentiation cultured with siFOXO3; PMA + CS + siFOXO3: neutrophils initiating NET differentiation cultured with CS ion solution and siFOXO3). The positive expression of NE or MPO and net structure DNA indicates that the neutrophils have differentiated into NETs.

Fig3. Pro-osteogenic implants inhibited NF-κB and NLRP3 signaling pathways accompanied with less NETs. (A-C) In neutrophil cluster of scRNA-seq, Gene Set Enrichment Analysis (GSEA) revealed the expression values of genes associated with NADP+1 - oxidoreductase activity, cell death in response to oxidative stress, and chromatin disassembly. (D) Volcano plot depicting the DEGs between various groups. Purple symbols indicate upregulated genes; green symbols indicate downregulated genes (log10 FDR < -1, log2 FC > 0.5). (E-G) Representative immunohistochemistry staining images and quantitative analysis of NE-positive and MPO-positive cells around implants at 3 days post-implantation. Scale bar: 100 μm, n=6. (H, I) Representative immunoblot images and semi-quantitative analysis of NE (H) and MPO (I) in neutrophils cultured on different implant materials for 12 h, n=6 independent experimental replicates. (J) Representative scanning electron microscopy (SEM) images of the NETosis (outlined by the white dotted line) induced by different surfaces at 4, 8, 12, and 24 h, respectively. Scale bar: 10 μm. (K) Represetnative immunofluorescent staining images and quantitative analysis of MPO (red), DNA component (SYTOX, green), and nucleus (blue) in neutrophils after 12 and 24 h. Scale bar:10 μm, n=6 independent experimental replicates. (L, M) Representative immunoblot images and semi-quantitative analysis of the phosphorylation levels in the NF-κB signaling pathway. (N) Transcriptional levels of genes associated with the NLRP3 signaling pathway, including Nlrp3, Asc, Caspase-1, Il-1β, and Il- 18. (O-S) Representative immunoblots and semi-quantitative analysis of the NLRP3 signaling pathway. n=6 independent experimental replicates. Data are means ± s.e.m. and ANOVA with Tukey’s post-hoc correction for multiple comparisons,

Figure 3 Abundant NET deposition in human GC tissues. (A) NETs were visualized in GC tissues as extracellular structures decorated with neutrophil elastase (green) and citrullinated H3 (red) co-localizing with DAPI/DNA (blue), but the lack of NETs in normal resection edge. Scale bar, 50 µm; magnification, ×400. (B) Western blot analysis results revealed that the level of cit-H3 was increased in the gastric cancer tissues but nearly absence in normal gastric tissues. The blots are representatives of 3 experiments from 10 pairs of patients with similar results. (C) The values of protein band densities were normalized to cit-h3 protein level of normal group. Data are shown as the means ± SEM, #P<0.001 vs. normal group. NET, neutrophil extracellular trap; GC, gastric cancer; cit-h3, citrullinated histone H3.

Figure 5.| Effects of NET inhibitors on solid tumor growth in nude mice.(E) Images showing representative immunostaining for neutrophil elastase (green), citrullinated histone H3 (red), and DAPI (blue) in the tumors of mice treated as indicated; scale bar, 50 µm; magnification, x400.