Journal: Nature Communications

Article Title: NR6A1 is essential for neural crest cell specification, formation and survival

doi: 10.1038/s41467-026-68647-2

Figure Lengend Snippet: A In situ hybridization of neuroepithelial markers (purple stain) in null and wildtype littermate control embryos at E9.0. Sox2 , Wnt1 and Pax3 are all expressed in the Nr6a1 null embryos, indicating the neural ectoderm has formed and patterned. B Immunostaining of SOX2 (green), PAX3 (green) and pHH3 (red) on transverse histological sections at E9.0. SOX2 and PAX3 are expressed in the neural plate in both null and control embryos. SOX2 expression is expanded to dorsal regions of the neural plate. PAX3 expression is maintained in the Nr6a1 null, although no PAX3 positive migratory cells could be observed further demonstrating the NCC deficiency. More pHH3 staining can be seen in the neural plate of the Nr6a1 null embryos suggesting the neural ectoderm is maintained in a highly proliferative state. A minimum of 5 embryos were assayed for each in situ hybridization marker and cell proliferation assay.

Article Snippet: The monoclonal antibodies SOX2 (R & D Systems, Minneapolis, MN) and pHH3 (Upstate/Millipore, Billerica, MA) were both used at 1:500.

Techniques: In Situ Hybridization, Staining, Control, Immunostaining, Expressing, Marker, Proliferation Assay

Journal: Frontiers in Molecular Biosciences

Article Title: The predictive role of protease-activated receptor (PAR-1) polymorphisms and activated microplatelets on the severity of atherosclerosis – preliminary studies

doi: 10.3389/fmolb.2025.1662954

Figure Lengend Snippet: The cytometric analysis (SSC and FCS) of isolated human resting platelets (A, D) , gated and labeled PAR-1 without activation (B, E) , and gated and labeled PAR-1 with activation by 10 µM TRAP (C, F) ; labeled with anti-CD61-FITC and PAR-1-APC antibodies (G–I) . The level of PAR-1 expression was read from gates P1. Markers M1 and M2 indicate the gates for microparticles and normal platelets, with the PAR-1 analysis applied to the summed population. An example image from a patient with DM is shown.

Article Snippet: Next: For the PAR-1 test without PLT activation, 5 μL of PAR-1-APC antibodies at a concentration of 5 μg/5 × 10 5 cells (Allophycocyanin (APC)-conjugated mouse monoclonal anti-human PAR-1; clone# 731115; mouse isotype: IgG1, R&D Systems, Minneapolis, Canada) and 5 μL of CD61-FITC antibodies (Monoclonal Mouse Anti-Human CD61, Platelet Glycoprotein IIIa/FITC, Clone Y2/51, code: F0803, DakoCytomation, Glostrup, Denmark) were added.

Techniques: Isolation, Labeling, Activation Assay, Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: The predictive role of protease-activated receptor (PAR-1) polymorphisms and activated microplatelets on the severity of atherosclerosis – preliminary studies

doi: 10.3389/fmolb.2025.1662954

Figure Lengend Snippet: The percentage of PAR-1 receptor expression before and after the addition of the thrombin receptor activating peptide (TRAP) in blood samples from patients with diabetic macroangiopathy (DM), the control group (CONTROL), and atherosclerosis obliterans (AO).

Article Snippet: Next: For the PAR-1 test without PLT activation, 5 μL of PAR-1-APC antibodies at a concentration of 5 μg/5 × 10 5 cells (Allophycocyanin (APC)-conjugated mouse monoclonal anti-human PAR-1; clone# 731115; mouse isotype: IgG1, R&D Systems, Minneapolis, Canada) and 5 μL of CD61-FITC antibodies (Monoclonal Mouse Anti-Human CD61, Platelet Glycoprotein IIIa/FITC, Clone Y2/51, code: F0803, DakoCytomation, Glostrup, Denmark) were added.

Techniques: Expressing, Control

Journal: Frontiers in Molecular Biosciences

Article Title: The predictive role of protease-activated receptor (PAR-1) polymorphisms and activated microplatelets on the severity of atherosclerosis – preliminary studies

doi: 10.3389/fmolb.2025.1662954

Figure Lengend Snippet: (A) Separation of DNA molecules in a 3% agarose gel of PAR-1 gene amplification products with the −506 I/D polymorphism. Lanes: 1 – homozygous I/I, 2 – heterozygous I/D, 3 – homozygous D/D, M–GeneRuler™ 50bp DNA Ladder (Fermentas). (B) The percentage distribution of the −506 I/D polymorphism variants in the PAR-1 gene: homozygous D/D (blue), heterozygous I/D (red), and homozygous I/I (green).

Article Snippet: Next: For the PAR-1 test without PLT activation, 5 μL of PAR-1-APC antibodies at a concentration of 5 μg/5 × 10 5 cells (Allophycocyanin (APC)-conjugated mouse monoclonal anti-human PAR-1; clone# 731115; mouse isotype: IgG1, R&D Systems, Minneapolis, Canada) and 5 μL of CD61-FITC antibodies (Monoclonal Mouse Anti-Human CD61, Platelet Glycoprotein IIIa/FITC, Clone Y2/51, code: F0803, DakoCytomation, Glostrup, Denmark) were added.

Techniques: Agarose Gel Electrophoresis, Amplification

Journal: Frontiers in Molecular Biosciences

Article Title: The predictive role of protease-activated receptor (PAR-1) polymorphisms and activated microplatelets on the severity of atherosclerosis – preliminary studies

doi: 10.3389/fmolb.2025.1662954

Figure Lengend Snippet: (A) The result of the restriction digestion of PCR products with the MvaI enzyme to check for the presence of the −1426 C/T polymorphism in the PAR-1 gene. Lanes: 1 – 6 homozygotes C/C, M–GeneRuler™ 100bp DNA Ladder (Fermentas). (B) The percentage distribution of the variants of the −1426 C/T polymorphism in the PAR-1 gene: homozygote C/C (blue color), heterozygote C/T (red color), homozygote T/T (green color).

Article Snippet: Next: For the PAR-1 test without PLT activation, 5 μL of PAR-1-APC antibodies at a concentration of 5 μg/5 × 10 5 cells (Allophycocyanin (APC)-conjugated mouse monoclonal anti-human PAR-1; clone# 731115; mouse isotype: IgG1, R&D Systems, Minneapolis, Canada) and 5 μL of CD61-FITC antibodies (Monoclonal Mouse Anti-Human CD61, Platelet Glycoprotein IIIa/FITC, Clone Y2/51, code: F0803, DakoCytomation, Glostrup, Denmark) were added.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: The predictive role of protease-activated receptor (PAR-1) polymorphisms and activated microplatelets on the severity of atherosclerosis – preliminary studies

doi: 10.3389/fmolb.2025.1662954

Figure Lengend Snippet: (A) Example separations of amplification products of the DNA fragment encompassing the IVSn-14 A/T polymorphism site of the PAR-1 gene using the SNaPshot method. Alleles were determined based on the size of primers and the colors of fluorescently labeled ddNTPs (terminators) incorporated during the primer extension reaction. (A) red peak, wild-type homozygote (AA); (B) green and red peaks, heterozygote (AT); (C) green peak, mutated homozygote (TT). (B) The percentage distribution of the variants of the IVS-14 A/T polymorphism of the PAR-1 gene is as follows: homozygote A/A (blue color), heterozygote A/T (red color), and homozygote T/T (green color).

Article Snippet: Next: For the PAR-1 test without PLT activation, 5 μL of PAR-1-APC antibodies at a concentration of 5 μg/5 × 10 5 cells (Allophycocyanin (APC)-conjugated mouse monoclonal anti-human PAR-1; clone# 731115; mouse isotype: IgG1, R&D Systems, Minneapolis, Canada) and 5 μL of CD61-FITC antibodies (Monoclonal Mouse Anti-Human CD61, Platelet Glycoprotein IIIa/FITC, Clone Y2/51, code: F0803, DakoCytomation, Glostrup, Denmark) were added.

Techniques: Amplification, Labeling

Journal: Frontiers in Molecular Biosciences

Article Title: The predictive role of protease-activated receptor (PAR-1) polymorphisms and activated microplatelets on the severity of atherosclerosis – preliminary studies

doi: 10.3389/fmolb.2025.1662954

Figure Lengend Snippet: Multivariate analysis: (A–D) Number of microparticles with PAR-1+TRAP; (E–F) Number of microparticles with BMI (Figure 4.12E-F) and age with smoking.

Article Snippet: Next: For the PAR-1 test without PLT activation, 5 μL of PAR-1-APC antibodies at a concentration of 5 μg/5 × 10 5 cells (Allophycocyanin (APC)-conjugated mouse monoclonal anti-human PAR-1; clone# 731115; mouse isotype: IgG1, R&D Systems, Minneapolis, Canada) and 5 μL of CD61-FITC antibodies (Monoclonal Mouse Anti-Human CD61, Platelet Glycoprotein IIIa/FITC, Clone Y2/51, code: F0803, DakoCytomation, Glostrup, Denmark) were added.

Techniques:

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: High-fat diet feeding increases cleaved TREM2 and TREM2 levels in gonadal white adipose tissue (A) Immunoblot analysis of TREM2 and cleaved TREM2 levels in gonadal white adipose tissue (GWAT), inguinal white adipose tissue (IWAT), and brown adipose tissue (BAT) of mice fed a high-fat diet (HFD) and normal chow diet (NCD) for 4 and 8 weeks ( n = 6 mice per group). (B) ELISA-based quantification of soluble TREM2 (sTREM2) levels in mouse serum ( n = 3 mice per group). (C) Schematic diagram illustrating cleaved membrane-bound TREM2 and soluble TREM2 forms. Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: Human/Mouse TREM2 Allophycocyanin Mab (FAB17291A, 1:50) were purchased from R&D systems.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Membrane

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: HFD feeding upregulates Adam10 and Adam17 expression in monocyte/macrophage populations of GWAT (A) Immunoblot analysis of ADAM10 and ADAM17 protein expression in gonadal white adipose tissue (GWAT) of mice after 8 weeks of NCD or HFD feeding ( n = 6 mice per group). (B) Umap plot of Adam10 , Adam17 , and total nuclei isolated from GWAT of NCD and HFD-fed mice (PRJNA942977). Clusters are colored by cell types: adipocyte, mesothelial cell, lymphatic endothelial cell, vascular endothelial cell, adipocyte progenitor cell, smooth muscle cell (SMC), monocyte/macrophage (mono/mac), dendritic cell (DC), B cell, and T cell. (C) Violin plot of Adam10 and Adam17 gene expression levels in total cell types from GWAT of NCD- and HFD-fed mice. (D) Umap plot of Adam10 , Adam17 from monocyte/macrophage population in GWAT of NCD- and HFD-fed mice (PRJNA942977). Clusters are colored by cell types: Lyve1 +macrophage (Mac. Lyve1 ), Trem2 +macrophage (Mac. Trem2 ), Prg4 +macrophage (Mac. Prg4 ), and monocyte. (E) Bubble plot and violin plot of Adam10 and Adam17 expression levels in distinct cell populations from mouse GWAT, determined by single-nucleus RNA sequencing (PRJNA942977). (F) qPCR analysis of Adam10 and Adam17 mRNA expression in isolated F4/80+ macrophages and adipocytes from GWAT after 8 weeks of NCD or HFD feeding ( n = 3 mice per group). Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: Human/Mouse TREM2 Allophycocyanin Mab (FAB17291A, 1:50) were purchased from R&D systems.

Techniques: Expressing, Western Blot, Isolation, Gene Expression, RNA Sequencing

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: Obesity increases ADAM10 and ADAM17 expression in macrophage populations of WAT (A) ADAM10 and ADAM17 expression levels in total cell types of human visceral adipose tissues ( GSE176171 ). Clusters are colored by cell types: Adipocyte, adipose stem and progenitor cells (ASPC), mesothelium, endothelial cell, lymphatic endothelial cell (LEC), pericyte, smooth muscle cell (SMC), macrophage, monocyte, dendritic cell (DC), mast cell, neutrophil, B cell, natural killer cell (NK cell), T cell, and endometrium cell. (B) Bubble plot and violin plot of ADAM10 and ADAM17 gene expressions and representative markers in adipocyte and macrophage populations with body mass index (BMI) in human subcutaneous adipose tissue ( GSE176171 ). (C) Violin plot of TREM2 , ADAM10 , and ADAM17 gene expression levels in human macrophage sub-populations (hMac1, hMac2, hMac3). (D) Correlation analysis of ADAM10 and ADAM17 gene expression levels with BMI in human subcutaneous adipose tissue ( n = 12 patients per group). Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: Human/Mouse TREM2 Allophycocyanin Mab (FAB17291A, 1:50) were purchased from R&D systems.

Techniques: Expressing, Gene Expression

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: Apoptotic adipocytes fail to induce TREM2 shedding in RAW264.7 cells and bone marrow-derived macrophages (BMDMs) (A) Schematic diagram illustrating the experimental method used for co-culturing dying/dead adipocytes with RAW264.7 cells or BMDMs. Apoptotic adipocytes (aAC) were generated by treating differentiated 3T3-L1 adipocytes with brefeldin A (BFA; 5 μg/mL for 24 h). Apoptotic adipocytes (1 × 10 5 cells/well) were directly co-cultured with RAW264.7 cells or BMDMs (5 × 10 5 cells/well) in growth medium. After 24 h of co-culture, non-engulfed or floating apoptotic adipocytes were removed by PBS washing, and RAW264.7 cells or BMDMs were subsequently harvested for immunoblot analysis. (B) Immunoblot analysis of caspase-3 expression levels in 3T3-L1 adipocytes after BFA for 24 h ( n = 3 cells per group). (C) and (D) Immunoblot analysis of TREM2, ADAM10, and ADAM17 protein expression levels in RAW264.7 cells (C) and BMDMs (D) co-cultured with apoptotic adipocytes ( n = 3 cells per group). Black arrow and red arrow indicate each precursor form of ADAM10/17 and active form of ADAM10/17. Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: Human/Mouse TREM2 Allophycocyanin Mab (FAB17291A, 1:50) were purchased from R&D systems.

Techniques: Derivative Assay, Generated, Cell Culture, Co-Culture Assay, Western Blot, Expressing

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: GM6001 blocks TREM2 shedding in RAW264.7 cells induced by pyroptotic adipocytes (A) A schematic diagram illustrating the experimental method used for co-culturing pyroptotic adipocytes with macrophages. Pyroptotic adipocytes were generated by treating differentiated 3T3L1 adipocytes with lipopolysaccharide (LPS; 100 μg/μL, 48 h) followed by ATP (2 mM, 24 h). Pyroptotic adipocytes (1 × 10 5 cells/well) were directly co-cultured with RAW264.7 cells (5 × 10 5 cells/well) or BMDMs for 24 h in growth medium. After co-culture, non-engulfed or floating pyroptotic adipocytes were removed by PBS washing, and RAW264.7 cells and BMDMs were subsequently harvested for immunoblot analysis. (B) Immunoblot analysis of NLRP3, caspase-1, and GSDMD (gasdermin D) expression levels in 3T3-L1 adipocytes after LPS priming for 48 h and ATP treatment for 24 h ( n = 3 cells per group). Black arrow and red arrow indicate each total form of GSDMD and cleaved GSDMD. (C) Immunoblot analysis of TREM2, ADAM10, and ADAM17 expression levels in RAW264.7 cells co-cultured with pyroptotic adipocytes for 24 h in the presence or absence of GM6001 ( n = 4 cells per group). Black arrow and red arrow indicate each precursor form of ADAM10/17 and active form of ADAM10/17. (D) Immunoblot analysis of P-STING and STING expression levels in RAW264.7 cells co-cultured with pyroptotic adipocytes ( n = 3 cells per group). (E) Immunoblot analysis of P-syk, syk, P-PI3K, PI3K, P-PLCγ1, PLCγ1, P-AKT, and AKT protein levels in RAW264.7 cells co-cultured with pyroptotic adipocytes ( n = 3 cells per group). (F) Phagocytosis analysis of RAW264.7 cells co-cultured with pyroptotic adipocytes for 18 h in the presence of GM6001. Adipocytes were tagged with C12-BODIPY (red), and macrophages were stained with DiO (green). Representative images from three independent experiments are shown, with quantification provided in the right panel. Scale bars, 100 μm. Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: Human/Mouse TREM2 Allophycocyanin Mab (FAB17291A, 1:50) were purchased from R&D systems.

Techniques: Generated, Cell Culture, Co-Culture Assay, Western Blot, Expressing, Staining

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: GM6001 prevents the cleavage of TREM2 on BMDMs induced by pyroptotic adipocytes (A) Immunoblot analysis of TREM2, ADAM10, and ADAM17 expression levels in BMDMs co-cultured with pyroptotic adipocytes for 24 h in the presence or absence of GM6001 ( n = 3 cells per group). Black arrow and red arrow indicate each precursor form of ADAM10/17 and active form of ADAM10/17. (B) and (C) Phagocytosis analysis of BMDMs, (B) TREM2 knockdown BMDMs, and negative control (NC). (C) co-cultured with pyroptotic adipocytes for 18 h in the presence of GM6001. Adipocytes were tagged with C12-BODIPY (red), and macrophages were stained with DiO (green). Representative images from three independent experiments are shown, with quantification provided in the right panel. The yellow boxes indicate the regions shown in the magnified images. Scale bars, 200 μm. Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: Human/Mouse TREM2 Allophycocyanin Mab (FAB17291A, 1:50) were purchased from R&D systems.

Techniques: Western Blot, Expressing, Cell Culture, Knockdown, Negative Control, Staining

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: GM6001 treatment reduces TREM2 shedding and HFD-induced inflammation in GWAT (A) Schematic illustration of the experimental strategy for GM6001 administration (7.5 mg/kg/2 days) in mice fed a high-fat diet (HFD) for 10 weeks. (B) Immunoblot analysis of TREM2, ADAM10, and ADAM17 protein levels in gonadal white adipose tissue (GWAT) of HFD-fed mice treated with or without GM6001 ( n = 4 mice per group). (C) Measurement of soluble TREM2 (sTREM2) levels in the serum of NCD- or HFD-fed mice treated with GM6001 for 10 weeks ( n = 3 mice per group). (D–F) Representative flow profile and quantification of (D) CD11B + CD45 + cells (E) M2/M1 macrophage ratio (CD206 + CD11C − CD45 + CD11B + /CD11C + CD206 − CD45 + CD11B + ), and (F) TREM2 + CD11C + and TREM2 + CD206 + macrophage populations in GWAT of GM6001-treated mice after feeding HFD for 10 weeks ( n = 4 mice per group). (G) Immunofluorescence staining of F4/80 (green) with DAPI (blue) counterstaining in paraffin-embedded GWAT sections from GM6001-treated and control mice ( n = 4 mice per group, scale bars, 100 μm). (H) Immunoblot analysis of P-STING, STING, NLRP3, F4/80, and caspase-1 expression levels in GWAT of HFD-fed mice treated with or without GM6001 ( n = 4 mice per group). Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: Human/Mouse TREM2 Allophycocyanin Mab (FAB17291A, 1:50) were purchased from R&D systems.

Techniques: Western Blot, Immunofluorescence, Staining, Control, Expressing