neutrophil elastase elane  (R&D Systems)

Journal: Cell death & disease

Article Title: ELANE enhances KEAP1 protein stability and reduces NRF2-mediated ferroptosis inhibition in metabolic dysfunction-associated fatty liver disease.

doi: 10.1038/s41419-025-07603-2

Figure Lengend Snippet: Fig. 2 Elane induces ferroptosis in the hepatocytes of MAFLD mice. A Primary mouse hepatocytes were extracted and treated with FFA (1 mM, OA:PA = 2:1) for 24 h with or without Elane (3 μg/mL), and the ROS levels in each group were assessed by flow cytometry (n = 3). B MDA levels in each group after FFA and/or Elane treatment (n = 3). C Flow cytometry was used to determine C11-BODIPY levels after FFA and/or Elane treatment (n = 3). D Western blotting for 4-hne protein levels after FFA and/or Elane treatment and grayscale analysis (n = 3). E Transmission electron microscopy was used to observe mitochondrial changes in cells after FFA and/or Elane treatment. F Cells were treated with the ferroptosis inhibitor Fer-1 (10 μM), and cell viability was assayed by a CCK-8 assay (n = 3). G Primary hepatocytes were extracted from Elane+/+ and Elane−/−mice after modelling, and ROS levels were assessed in each group by flow cytometry (n = 3). H MDA levels in primary hepatocytes after modelling in Elane+/+ and Elane−/−mice (n = 3). I Flow cytometry analysis of C11-BODIPY levels in primary hepatocytes after modelling in Elane+/+ and Elane-/- mice (n = 3). J Transmission electron microscopy was used to observe mitochondrial changes in primary hepatocytes after modelling in Elane+/+ and Elane−/−mice. Scale bars: 500 nm. The data are presented as the means ± SDs. *P < 0.05, **P < 0.01, in comparison to the control group; ns not significant.

Article Snippet: The levels of Elane in the tissue homogenate and plasma from different groups were quantified with a mouse Elane ELISA kit (E-EL-M3025, Elabscience) following the manufacturer’s instructions.

Techniques: Cytometry, Flow Cytometry, Western Blot, Transmission Assay, Electron Microscopy, CCK-8 Assay, Comparison, Control

Journal: Cell death & disease

Article Title: ELANE enhances KEAP1 protein stability and reduces NRF2-mediated ferroptosis inhibition in metabolic dysfunction-associated fatty liver disease.

doi: 10.1038/s41419-025-07603-2

Figure Lengend Snippet: Fig. 3 Elane regulates ferroptosis in hepatocytes via Gpx4. A Immunohistochemistry staining was used to assess Gpx4 expression in liver tissue after modelling in Elane+/+ and Elane−/−mice (n = 3). Scale bars: 100 μm. B, C Western blotting was used to assess Gpx4 protein expression in liver tissues after modelling in Elane+/+ and Elane−/−mice, and a graded analysis was performed (n = 3). D qRT‒PCR was used to assess the mRNA expression of Gpx4 in the liver tissues of Elane+/+ and Elane−/−mice after modelling (n = 3). E Extraction of protein from primary mouse hepatocytes, followed by Western blotting to assess Gpx4 protein levels after FFA and/or Elane treatment, after which grayscale analysis was performed (n = 3). F, G Western blotting was used to assess Gpx4 protein levels after the overexpression of Gpx4 by FFA and/or Elane treatment, after which grayscale analysis was performed (n = 3). H MDA levels after the overexpression of Gpx4 following FFA and/or Elane treatment (n = 3). I CCK-8 was used to assess cell viability after the overexpression of Gpx4 after FFA and/or Elane treatment (n = 3). J, K Extraction of protein from primary hepatocytes from Elane+/+ and Elane−/−mice after modelling, followed by Western blotting to assess Gpx4 protein expression, after which grayscale analysis was performed (n = 3). L MDA levels in primary hepatocytes from Elane+/+ and Elane−/−mice after modelling were assessed after 24 h of treatment with the Gpx4 inhibitor RSL3 (2 μM) (n = 3). M Cell viability of primary hepatocytes after modelling in Elane+/+ and Elane−/−mice was assayed by CCK-8 after the addition of RSL3 for 24 h (n = 3). The data are presented as the means ± SDs. *P < 0.05, **P < 0.01, in comparison to the control group; ns not significant.

Article Snippet: The levels of Elane in the tissue homogenate and plasma from different groups were quantified with a mouse Elane ELISA kit (E-EL-M3025, Elabscience) following the manufacturer’s instructions.

Techniques: Immunohistochemistry, Staining, Expressing, Western Blot, Extraction, Over Expression, CCK-8 Assay, Comparison, Control

Journal: Cell death & disease

Article Title: ELANE enhances KEAP1 protein stability and reduces NRF2-mediated ferroptosis inhibition in metabolic dysfunction-associated fatty liver disease.

doi: 10.1038/s41419-025-07603-2

Figure Lengend Snippet: Fig. 5 Elane inhibits the degradation of Keap1. A qRT‒PCR was used to assess Keap1 mRNA expression in primary hepatocytes after FFA and/or Elane treatment (n = 3). B qRT‒PCR was used to assess the mRNA half-life of Keap1 after FFA and/or Elane treatment of primary hepatocytes (n = 3). C, D Western blotting was used to assess the protein half-life of Keap1 after FFA and/or Elane treatment of primary hepatocytes (n = 3). E qRT‒PCR was used to assess the mRNA expression of Keap1 in primary hepatocytes after modelling in Elane+/+ and Elane−/−mice (n = 3). F qRT‒PCR was used to assess the mRNA half-life of Keap1 in primary hepatocytes after modelling in Elane+/+ and Elane−/−mice (n = 3). G, H Western blotting was used to assess the protein half-life of Keap1 in primary hepatocytes after modelling in Elane+/ + and Elane−/−mice (n = 3). I, J After MG132 (10 μM) was added for 4 h, Western blotting was performed to assess the protein expression of Keap1 after FFA and/or Elane treatment, after which grayscale analysis was performed (n = 3). K, L After the addition of CQ (20 μM) for 8 h, Western blotting was performed to assess the protein expression of Keap1 after FFA and/or Elane treatment, after which grayscale analysis was performed (n = 3). The data are presented as the means ± SDs. *P < 0.05, **P < 0.01, in comparison to the control group; ns not significant.

Article Snippet: The levels of Elane in the tissue homogenate and plasma from different groups were quantified with a mouse Elane ELISA kit (E-EL-M3025, Elabscience) following the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Comparison, Control

Journal: Cell death & disease

Article Title: ELANE enhances KEAP1 protein stability and reduces NRF2-mediated ferroptosis inhibition in metabolic dysfunction-associated fatty liver disease.

doi: 10.1038/s41419-025-07603-2

Figure Lengend Snippet: Fig. 6 Elane attenuates P62 binding to Keap1 and increases Keap1 stability. A qRT‒PCR analysis of P62 mRNA expression in primary hepatocytes after FFA and/or Elane treatment (n = 3). B Western blotting for protein expression and grayscale analysis of P62 in primary hepatocytes after FFA and/or Elane treatment (n = 3). C qRT‒PCR was used to assess the mRNA expression of P62 in primary hepatocytes after modelling in Elane+/+ and Elane−/−mice (n = 3). D Western blotting for protein expression and grayscale analysis of P62 in primary hepatocytes after modelling in Elane+/+ and Elane−/−mice (n = 3). E, F Proteins were immunoprecipitated with Keap1 or P62 antibodies, and the binding of Keap1 and P62 proteins was assessed after FFA and/or Elane treatment of primary hepatocytes. G, H Binding of Keap1 and P62 proteins in primary hepatocytes after Elane+/+ and Elane−/−mouse models were established was assessed by immunoprecipitating the proteins with Keap1 or P62 antibodies. The data are presented as the means ± SDs. *P < 0.05, **P < 0.01, in comparison to the control group; ns not significant.

Article Snippet: The levels of Elane in the tissue homogenate and plasma from different groups were quantified with a mouse Elane ELISA kit (E-EL-M3025, Elabscience) following the manufacturer’s instructions.

Techniques: Binding Assay, Expressing, Western Blot, Immunoprecipitation, Comparison, Control

Journal: Journal of hepatology

Article Title: Ductular reaction-associated neutrophils promote biliary epithelium proliferation in chronic liver disease

doi: 10.1016/j.jhep.2023.05.045

Figure Lengend Snippet: (A) Immunofluorescence of MPO and citH3 in WT and Pad4 −/− liver sections (n = 4 mice per group). Scale bars: 100 μm. (B) Immunohistochemistry analysis of WT and Pad4 −/− mice liver sections (n = 4–7 mice per group). Scale bars: 100 μm. (C) Immunofluorescence of KRT19-EpCAM and KRT19-SOX9 in WT and Pad4 −/− mouse liver sections. Scale bars: 100 μm. (D) Immunofluorescence of KRT19 and CCND1 in WT and Pad4 −/− mice (n = 4–6 mice per group). Scale bars: 50 μm. (E) Immunofluorescence of NETs in WT and Elane −/− liver sections (n = 4 mice per group). Scale bars: 100 μm. (F) Immunohistochemistry analysis of MPO and KRT19 in WT and Elane −/− mouse liver sections (n = 4–5 mice per group). Scale bars: 100 μm. (G) Immunofluorescence of progenitor markers in WT and Elane −/− mouse liver sections. Scale bars: 100 μm. (H) Immunofluorescence of KRT19 and CCND1 in WT and Elane −/− mice. Scale bars: 50 μm. (I) Images of biliary organoids and WT or Elane −/− neutrophil co-culture. Organoid area is normalized per microcavity area. Each measurement represents a technical replicate consisting of the average area of at least 40 microcavities (n = 3 mice-derived organoids). Scale bar: 500 μm. Data presented as mean ± SEM. * p <0.05, ** p <0.01 and *** p <0.001 as determined by Student’s t test (A, B, D, E, F, H) and one-way ANOVA with Tukey’s multiple comparison test (I). CCND1, cyclin D1; citH3, citrullinated histone H3; DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine; Elane, neutrophil elastase; EpCAM, epithelial cell adhesion molecule; FOV, field of view; KRT, cytokeratin; MPO, myeloperoxidase; NETs, neutrophil extracellular traps; Pad4, peptidyl arginine deiminase 4 ; SOX9, SRY-Box transcription factor 9; WT, wild-type.

Article Snippet: Neutrophil elastase- and protein arginine deiminase 4-deficient mice ( Elane −/− and Pad4 −/− ) were obtained from Jackson Laboratory.

Techniques: Immunofluorescence, Immunohistochemistry, Co-Culture Assay, Derivative Assay, Comparison

Journal: Cell death discovery

Article Title: Highly sensitive magnetic particle imaging of abdominal aortic aneurysm NETosis with anti-Ly6G iron oxide nanoparticles.

doi: 10.1038/s41420-024-02156-3

Figure Lengend Snippet: Fig. 2 Neutrophil infiltration and NETs release within AAA lesions. A IF staining for neutrophil marker (Ly6G) was performed on tissue sections of the mouse abdominal aorta. B Hyalinization and IF labeling of the whole abdominal aorta and 3D tissue imaging were acquired with light-sheet microscopy. C–E Quantification of NETs markers (ELANE and MPO) in mouse abdominal aorta by western blotting (n = 3). **P < 0.01, ****P < 0.0001, indicating statistical significance between the two groups.

Article Snippet: Protein expression was detected using primary antibodies against ELANE (Boster Biotech, Wuhan, China), MPO (Proteintech, Wuhan, China) and GAPDH (Servicebio, Wuhan, China).

Techniques: Staining, Marker, Labeling, Imaging, Microscopy, Western Blot