55549 rrid ab 831596  (Santa Cruz Biotechnology)

Journal: Advanced Science

Article Title: LRG1 Drives Pathological Angiogenesis by Disrupting Neutrophil Mitochondrial Homeostasis in Bladder Cancer

doi: 10.1002/advs.76604

Figure Lengend Snippet: Genetic ablation of Lrg1 restrains tumor progression, normalizes vasculature, and reshapes the immune microenvironment. (A) Tumor growth curves, macroscopic anatomical images, and endpoint tumor weights of wild‐type (LRG1 +/+ ) and LRG1 knockout (LRG1 −/− ) subcutaneous MB49 tumor models. (B, C) Representative bioluminescence images (B) and macroscopic anatomical observations (C) of orthotopic bladder tumors in LRG1 +/+ and Lrg1 −/− mice. (D, E) Representative bioluminescence images with corresponding statistical analysis (D), and macroscopic anatomical images of the lungs detailing metastatic nodules along with their quantification (E) in the experimental tail‐vein injection model. (F) Immunofluorescence staining for CD31 and quantitative analysis of overall microvessel density and vessel diameter. (G, H) Representative confocal double immunofluorescence images for CD31 (green) and α‐SMA (red) (G) in subcutaneous tumor models and quantitative analysis of pericyte coverage (H) in both subcutaneous and orthotopic tumor models. (I) Scanning electron microscopy (SEM) images displaying the ultrastructure of tumor vascular endothelium and intraluminal membranous inclusions. (J) Expression profiles of canonical marker genes across different cell clusters, with red indicating high expression and blue indicating low expression. (K) Uniform Manifold Approximation and Projection (UMAP) plot displaying the distribution of major cell lineages in the mouse BCa tumor tissues, color‐coded by distinct clusters. (L) UMAP plot demonstrating the subclustering of neutrophils into four distinct subpopulations (C1‐C4). (M) UCell functional enrichment scoring of the NETs signature gene set within the neutrophil populations, where brighter colors represent higher enrichment scores. (N) Violin plots quantifying the NETs gene set scores across the neutrophil subpopulations. (O) Comparative relative proportions of the four neutrophil subpopulations between the LRG1‐OE and WT groups.

Article Snippet: Serum concentrations of LRG1 (RK01800, ABclonal, China), as well as the NETosis‐associated markers Myeloperoxidase (MPO; RK00310, ABclonal, China) and Neutrophil Elastase (NE; RK00694, ABclonal, China), were determined using commercial ELISA kits.

Techniques: Knock-Out, Injection, Immunofluorescence, Staining, Electron Microscopy, Expressing, Marker, Functional Assay

Journal: Advanced Science

Article Title: LRG1 Drives Pathological Angiogenesis by Disrupting Neutrophil Mitochondrial Homeostasis in Bladder Cancer

doi: 10.1002/advs.76604

Figure Lengend Snippet: Tumor‐derived LRG1 promotes cancer progression by recruiting neutrophils and driving NETosis. (A, B) Representative IHC images and quantification of CD66b+ neutrophil infiltration density in HM+ versus HM‐ BCa tissues. (C) Transwell assays evaluating the chemotactic effect of tumor‐derived LRG1 on isolated human neutrophils. (D) Representative macroscopic images and endpoint tumor weights demonstrating the “neutrophil‐dependent” tumor‐promoting effect of LRG1 in vivo, which is effectively rescued by anti‐Ly6G antibody depletion. (E) UMAP plot demonstrating the reclustering and annotation of distinct neutrophil subpopulations based on subcluster‐specific marker genes from the scRNA‐seq cohort. (F) Gene set enrichment scoring of the KEGG NETs pathway across neutrophil subpopulations, highlighting significant enrichment in the NCF2_neu subset. (G) Comparative analysis of the relative proportion of the NCF2_neu subpopulation between normal and BCa tumor tissues. (H) GSVA correlation analysis between the LRG1_tumor meta‐program and NCF2_neu infiltration abundance across three independent bulk RNA‐seq datasets. (I) ELISA quantification of serum NETs markers (cfDNA, MPO, NE) and their correlation with LRG1 levels. (J) Immunofluorescence staining for MPO and H3Cit in neutrophils directly stimulated with rh‐LRG1 or PMA. (K) Representative macroscopic images and endpoint tumor weights demonstrating the rescue of LRG1‐induced tumor progression by DNase I treatment in vivo.

Article Snippet: Serum concentrations of LRG1 (RK01800, ABclonal, China), as well as the NETosis‐associated markers Myeloperoxidase (MPO; RK00310, ABclonal, China) and Neutrophil Elastase (NE; RK00694, ABclonal, China), were determined using commercial ELISA kits.

Techniques: Derivative Assay, Isolation, In Vivo, Marker, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: LRG1 Drives Pathological Angiogenesis by Disrupting Neutrophil Mitochondrial Homeostasis in Bladder Cancer

doi: 10.1002/advs.76604

Figure Lengend Snippet: NETs facilitate tumor cell trapping and directly destabilize tumor vasculature. (A) In vitro neutrophil adhesion assay evaluating the trapping effect of NETs on BCa cells and the reversal by DNase I. (B) Transwell co‐culture assay evaluating the impact of intact NETs on the migratory capacity of BCa cells. (C) In vitro tube formation assay utilizing HUVECs to assess the pro‐angiogenic capacity of NETs. (D) Endothelial permeability assay measuring TRITC‐Dextran flux across HUVEC monolayers following NETs stimulation. (E) Schematic timeline of the in vivo LPS‐induced systemic NETosis model and DNase I intervention. (F, G) Endpoint tumor weights (F) and macroscopic images (G) of subcutaneous tumors from respective groups. (H, I) Representative immunofluorescence images depicting neutrophil recruitment (Ly6G, H) and NETs formation (H3Cit, I) in tumor tissues. (J, K) Quantitative analysis of mural cell coverage (J) and representative confocal dual immunofluorescence images for CD31/α‐SMA (K) to confirm NET‐induced vascular destabilization.

Article Snippet: Serum concentrations of LRG1 (RK01800, ABclonal, China), as well as the NETosis‐associated markers Myeloperoxidase (MPO; RK00310, ABclonal, China) and Neutrophil Elastase (NE; RK00694, ABclonal, China), were determined using commercial ELISA kits.

Techniques: In Vitro, Cell Adhesion Assay, Co-culture Assay, Tube Formation Assay, Permeability, In Vivo, Immunofluorescence

Journal: Advanced Science

Article Title: LRG1 Drives Pathological Angiogenesis by Disrupting Neutrophil Mitochondrial Homeostasis in Bladder Cancer

doi: 10.1002/advs.76604

Figure Lengend Snippet: LRG1 directly interacts with ANXA2 via its LRR domain and impedes the mitochondrial translocation of active Akt. (A) Silver staining and Venn diagram of mass spectrometry analysis identifying ANXA2 as a direct interactor following pull‐down assays with anti‐LRG1 or anti‐His antibodies. (B) Structural binding mode diagram illustrating the molecular interaction interface between LRG1 (yellow) and ANXA2 (blue). (C, D) Endogenous (C, neutrophils) and exogenous (D, HEK‐293T) Co‐IP assays validating the physical interaction between LRG1 and ANXA2. (E) Flow cytometric validation of CD11b expression in DMSO‐differentiated HL‐60 (dHL‐60) cells. (F) Confocal microscopy demonstrating the colocalization of LRG1 and ANXA2 in the cytoplasm of primary neutrophils and dHL‐60 cells. (G, H) Schematic of LRG1 truncation mutants (G) and Co‐IP assays (H) pinpointing the LRR1‐8 domain as essential for ANXA2 binding. (I) qRT‐PCR and Western blot analysis of ANXA2 expression following LRG1 stimulation. (J) Immunofluorescence confirming the colocalization of ANXA2 with the mitochondrial marker TOM20. (K, L) Immunoblotting of isolated mitochondrial fractions showing the suppressive effect of LRG1 on ANXA2 phosphorylation (K) and the concurrent reduction of mitochondrial pSer473‐Akt (L).

Article Snippet: Serum concentrations of LRG1 (RK01800, ABclonal, China), as well as the NETosis‐associated markers Myeloperoxidase (MPO; RK00310, ABclonal, China) and Neutrophil Elastase (NE; RK00694, ABclonal, China), were determined using commercial ELISA kits.

Techniques: Translocation Assay, Silver Staining, Mass Spectrometry, Binding Assay, Co-Immunoprecipitation Assay, Biomarker Discovery, Expressing, Confocal Microscopy, Quantitative RT-PCR, Western Blot, Immunofluorescence, Marker, Isolation, Phospho-proteomics

Journal: Journal of Clinical and Translational Hepatology

Article Title: Single-cell Sequencing Reveals Neutrophil Extracellular Traps in Association with Endotheliopathy and Immunothrombosis in Hepatitis B Virus-related Acute-on-chronic Liver Failure

doi: 10.14218/JCTH.2025.00666

Figure Lengend Snippet: (A) Plasma concentrations of NETs markers, including MPO–DNA complexes, MPO, and NE, in HBV-ACLF, CHB, and HC groups. (B–C) Correlations between plasma MPO–DNA levels and INR and TAT (B), and LDH (C). (D) Correlations between plasma MPO–DNA levels and established prognostic scoring systems, including the COSSH-ACLF II and MELD-Na scores. (E) Comparison of NETs marker levels (MPO–DNA and MPO) between survivors and non-survivors within the HBV-ACLF cohort. (F) Kaplan–Meier survival curves of HBV-ACLF patients stratified by plasma MPO–DNA levels (≤2-fold vs >2-fold). *** P < 0.001, and **** P < 0.0001. NETs, neutrophil extracellular traps; HBV-ACLF, hepatitis B virus-related acute-on-chronic liver failure; MPO, myeloperoxidase; NE, neutrophil elastase; CHB, chronic hepatitis B; HC, healthy controls; INR, international normalized ratio; TAT, thrombin-antithrombin complex; LDH, lactate dehydrogenase; COSSH-ACLF II, Chinese Onset Study of Severe Hepatitis with Acute-on-Chronic Liver Failure II; MELD-Na, Model for End-Stage Liver Disease-Sodium.

Article Snippet: Specifically, the Human Myeloperoxidase ELISA Kit (RayBiotech, Cat. No. ELH-MPO) and the Human Neutrophil Elastase ELISA Kit (RayBiotech, Cat. No. ELH-NEUTRO-1) were used according to the manufacturers’ protocols.

Techniques: Clinical Proteomics, Comparison, Marker, Virus

Journal: Journal of Clinical and Translational Hepatology

Article Title: Single-cell Sequencing Reveals Neutrophil Extracellular Traps in Association with Endotheliopathy and Immunothrombosis in Hepatitis B Virus-related Acute-on-chronic Liver Failure

doi: 10.14218/JCTH.2025.00666

Figure Lengend Snippet: (A–C) Correlation analyses between plasma MPO–DNA complex levels and syndecan-1 (A), ICAM-1 (B), and sTM and vWF (C). (D) Immunofluorescence staining of endothelial cells (CD31), NETs (citrullinated histone H3, Cit-H3), and platelets (CD41) in liver tissues from patients with HBV-ACLF, CHB, and HC. Scale bars: 50 µm. (E) Neutrophils isolated from CHB patients were stimulated with PMA. NETs were collected and incubated with human aortic endothelial cells, with or without DNase I treatment. Syndecan-1 levels in supernatants and plasma clotting time of cells were measured after 6 h. ** P < 0.01, *** P < 0.001, and **** P < 0.0001. NETs, neutrophil extracellular traps; HBV-ACLF, hepatitis B virus-related acute-on-chronic liver failure; MPO, myeloperoxidase; ICAM-1, intercellular adhesion molecule-1; sTM, soluble thrombomodulin; vWF, von Willebrand factor; CHB, chronic hepatitis B; HC, healthy controls; PMA, phorbol 12-myristate 13-acetate; DNase I, deoxyribonuclease I.

Article Snippet: Specifically, the Human Myeloperoxidase ELISA Kit (RayBiotech, Cat. No. ELH-MPO) and the Human Neutrophil Elastase ELISA Kit (RayBiotech, Cat. No. ELH-NEUTRO-1) were used according to the manufacturers’ protocols.

Techniques: Clinical Proteomics, Immunofluorescence, Staining, Isolation, Incubation, Coagulation, Virus