Journal: International Journal of Biological Sciences

Article Title: N-acetylcysteine and raloxifene boost photodynamic therapy against cutaneous squamous cell carcinoma by decreasing TGFβ1 secreted by cancer-associated fibroblasts

doi: 10.7150/ijbs.106642

Figure Lengend Snippet: Effect of CAF endoglin expression levels on TGFβ1 secretion and on the induction of resistance to PDT in A431 cells. (A) Secretion of TGFβ1 produced by T205A after treatment with increasing concentrations of recombinant endoglin. ELISA assay was performed to quantify the levels of secreted TGFβ1 to the culture medium collected 24 h after treatments. (B) Expression of endoglin analyzed by Western blot in T205A CAFs after transfection with the negative control (NC) and a siRNA targeting endoglin. Western blot images are representative of n = 3 independent experiments. Error bars denote ± S.E.M. (n = 4, one-way ANOVA: *p<0.05, **p<0.01 and ***p<0.001). (C) Secretion of TGFβ1 produced by T205A CAFs after transfection with NC or ENGsiRNA. ELISA assay was performed to quantify the levels of secreted TGFβ1 to the culture medium collected 24 h after treatment with NAC (D) Effect of the pre-treatment with NAC of transfected T205A in the CAF-derived CM on the response of bidimensional A431 cell cultures to PDT. Upper row: phase contrast images illustrating the morphological changes in A431 cells after treatments. Scale bar: 50 µm. Lower row: cell viability rates of A431 cells treated with MAL-PDT (0.5 mM of MAL and red light dose of 9.1 J·cm -2 ) in the presence of T205A CM after transfection. Error bars denote ± S.E.M. (n = 3, one-way ANOVA, statistical comparisons to Fb condition: *<0.05, **p<0.01 and ***p<0.001; multiple comparisons: #p<0.05, ##p<0.01 and ###p<0.001).

Article Snippet: Lyophilized recombinant human endoglin (R&D Systems, Minneapolis, MN, USA) was reconstituted in PBS containing 0.1% BSA to prepare a 250 μg/ml stock solution.

Techniques: Expressing, Produced, Recombinant, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Negative Control, Derivative Assay

Journal: Cells

Article Title: Microglial Phenotyping in Neurodegenerative Disease Brains: Identification of Reactive Microglia with an Antibody to Variant of CD105/Endoglin

doi: 10.3390/cells8070766

Figure Lengend Snippet: Details of antibodies used in study.

Article Snippet: CD105 antibodies from R&D Systems (Minneapolis, MN, USA) (MAB1097) or Abcam (Cambridge, MA, USA.) (AB221675) were mixed for 18 hours with recombinant CD105 protein (1097-EN, R&D Systems), which consists of amino acids 26–586 of human CD105 expressed in mouse myeloma cells.

Techniques:

Journal: Cells

Article Title: Microglial Phenotyping in Neurodegenerative Disease Brains: Identification of Reactive Microglia with an Antibody to Variant of CD105/Endoglin

doi: 10.3390/cells8070766

Figure Lengend Snippet: Patterns of Staining of CD105/MAB1097 in human brain sections. ( a ) Staining of parallel sections of MTG from a low-plaque ND case for CD105/MAB1097 (panel A) (CD105/RnD) and IBA-1 (panel B). Staining of parallel sections of MTG from an AD case for CD105/RnD (panel C) or IBA-1 (panel D). All images have the same magnification. Scale bars represent 50 μm. ( b ) Low magnification images showing distribution of CD105-positive cells in low-plaque ND (panel A) and AD cases (panel B). Representative positive cells indicated by purple arrows. Both images at same magnification. Scale bars represent 100 μm. ( c ) Representative image of CD105 positive cells in low-plaque ND (LP) (panel A) (purple arrow) Morphology of CD105-positive microglia compared to IBA-1-positive surrounding cells (brown arrow) in LP case (panel B). Images of CD105-positive microglia with activated morphology in HP (panel C) and AD cases (panel D) (purple arrows). (panel E) Vascular staining in AD case (purple arrow) using CD105/Thermo Fisher antibody. All images at same magnification. Scale bars represent 50 μm.

Article Snippet: CD105 antibodies from R&D Systems (Minneapolis, MN, USA) (MAB1097) or Abcam (Cambridge, MA, USA.) (AB221675) were mixed for 18 hours with recombinant CD105 protein (1097-EN, R&D Systems), which consists of amino acids 26–586 of human CD105 expressed in mouse myeloma cells.

Techniques: Staining

Journal: Cells

Article Title: Microglial Phenotyping in Neurodegenerative Disease Brains: Identification of Reactive Microglia with an Antibody to Variant of CD105/Endoglin

doi: 10.3390/cells8070766

Figure Lengend Snippet: Biochemical Characterization of CD105 antibody MAB1097-detected polypeptides. ( a ) CD105 antibody MAB1097 (CD105/RnD)-identified polypeptides in protein samples of control unstimulated (CON), and polyIC (pIC) and Aβ-stimulated human brain-derived microglia (MG), and brain-derived endothelial cells (EC). ( b ) CD105 antibody MAB1097 (CD105/RnD)-identified polypeptides in protein samples of brain from middle temporal gyrus of high-plaque (HP), Alzheimer’s disease (AD) and low-plaque non-demented (ND) cases. ( c ) Antibody absorption studies. (Left hand panels): Images of Western blots containing brain MTG samples (LP, AD) and cell samples from THP-1-derived macrophages (MAC) reacted with CD105 antibody MAB1097 (RnD) preabsorbed with CD105 peptide (+Pep) or without peptide (-Pep). (Right hand panels): Images of Western blots of cell samples from THP-1-derived macrophages (MAC) or brain endothelial cell line hCMEC/D3 (EC) reacted with CD105 antibody AbCAM (AB221675) preabsorbed with CD105 peptide (+Pep) or without peptide (−Pep). ( d ) CD105 immunoreactive polypeptides after deglycosylation. Images of Western blots with CD105 antibodies MAB1097 (RnD), AB221675 (AbCAM) and PA5-32303 (Thermo) for two separate AD brain-derived protein extracts (AD1 and AD2) treated with PNGase F enzyme (+) or without enzyme (−). ( e ) Immunoprecipitation with CD105 antibodies. Images of Western blots with samples of brain (AD1 and AD2) or THP-1-derived macrophages (MAC) samples immunoprecipated with CD105 antibodies AbCAM or Thermo Fisher, and detected with CD105/RnD. MAC-T: sample of total protein of THP-1 macrophages.

Article Snippet: CD105 antibodies from R&D Systems (Minneapolis, MN, USA) (MAB1097) or Abcam (Cambridge, MA, USA.) (AB221675) were mixed for 18 hours with recombinant CD105 protein (1097-EN, R&D Systems), which consists of amino acids 26–586 of human CD105 expressed in mouse myeloma cells.

Techniques: Control, Derivative Assay, Western Blot, Immunoprecipitation

Journal: Cells

Article Title: Microglial Phenotyping in Neurodegenerative Disease Brains: Identification of Reactive Microglia with an Antibody to Variant of CD105/Endoglin

doi: 10.3390/cells8070766

Figure Lengend Snippet: Absorption controls for CD105 staining of microglia and antibody characterization. ( a ) Antibody absorption. (panels A–D). Staining of sections from AD cases (AD1) (A and B) or AD2 with CD105 antibody preabsorbed with immunizing peptide (+Pep) compared to staining of matched sections with CD105 antibody non-absorbed (−Pep). All images are at same magnification: scale bars represent 50 μm. ( b ) Deglycosylation. Tissue section (AD case) was treated with PNGase F in solution (+PNGase F) (panel A) or underwent control treatment without enzyme (−PNGase F) (panel B), and then reacted with CD105/RnD antibody. Images are at same magnification: scale bars represent 50 μm. ( c ) Antigen retrieval. Tissue sections from an AD case underwent antigen retrieval procedure prior to immunocytochemistry with antibodies CD105/RnD (panel A) or CD105/AbCAM (panel B). The position of an unstained large vessel is outlined (panel A). Vessel staining indicated with purple arrows (panel B). Scale bars represent 50 μm.

Article Snippet: CD105 antibodies from R&D Systems (Minneapolis, MN, USA) (MAB1097) or Abcam (Cambridge, MA, USA.) (AB221675) were mixed for 18 hours with recombinant CD105 protein (1097-EN, R&D Systems), which consists of amino acids 26–586 of human CD105 expressed in mouse myeloma cells.

Techniques: Staining, Control, Immunocytochemistry

Journal: Cells

Article Title: Microglial Phenotyping in Neurodegenerative Disease Brains: Identification of Reactive Microglia with an Antibody to Variant of CD105/Endoglin

doi: 10.3390/cells8070766

Figure Lengend Snippet: Images demonstrating colocalization of CD105/MAB1097 with microglial markers ( a ) panels A, B Low magnification images of high-plaque (HP) case (panel A) and AD case (panel B) showing staining with CD105/MAB1097 (purple arrows) and IBA-1 (brown arrows) in MTG sections. Both images at same magnification: scale bars represent 100 μm. ( b ) (panel A) CD105 microglia (purple arrows) with ramified morphology stained with antibody to P2RY12 (brown arrow) in low-plaque (LP) case. (panel B) CD105 microglia (purple arrows) and HLA-DR (brown arrow) in AD case. (panel C) CD105 microglia (purple arrow) and CD45 (brown arrow) in AD case. All sections were from MTG. Images at same magnification: scale bars represent 50 μm. ( c ) (panel A–E). Patterns of staining of CD105 (purple) and IBA-1 (brown) in MTG sections of cases with progressively increasing pathology from LP to AD. (panel F). CD105-positive microglia in hippocampus (HPC) of AD case. Images at same magnification: scale bars represent 50 μm.

Article Snippet: CD105 antibodies from R&D Systems (Minneapolis, MN, USA) (MAB1097) or Abcam (Cambridge, MA, USA.) (AB221675) were mixed for 18 hours with recombinant CD105 protein (1097-EN, R&D Systems), which consists of amino acids 26–586 of human CD105 expressed in mouse myeloma cells.

Techniques: Staining

Journal: Cells

Article Title: Microglial Phenotyping in Neurodegenerative Disease Brains: Identification of Reactive Microglia with an Antibody to Variant of CD105/Endoglin

doi: 10.3390/cells8070766

Figure Lengend Snippet: Different patterns of colocalization of CD105/MAB1097 and IBA-1 in microglia in AD cases by confocal microscopy. ( a ) IBA-1-positive (green) microglia; CD105-positive immunoreactive cells (red); Merged image showing colocalized CD105 and IBA-1 (yellow). Blue identifies nuclei revealed by staining with DAPI. Scale bars represent 50 μm. ( b ) Higher magnification image of a single cell with activated morphology. IBA-1-positive (green) microglia, strongly staining CD105-positive microglia with activated morphology (red). Merged image showing colocalized signals (yellow) within cell. Scale bars represent 25 μm. ( c ) Images to show different amounts of CD105 expression in microglia in an AD case. CD105 immunoreactivity (green), IBA-1 immunoreactivity (red), and merged image (yellow). Scale bars represent 50 μm.

Article Snippet: CD105 antibodies from R&D Systems (Minneapolis, MN, USA) (MAB1097) or Abcam (Cambridge, MA, USA.) (AB221675) were mixed for 18 hours with recombinant CD105 protein (1097-EN, R&D Systems), which consists of amino acids 26–586 of human CD105 expressed in mouse myeloma cells.

Techniques: Confocal Microscopy, Staining, Expressing

Journal: Cells

Article Title: Microglial Phenotyping in Neurodegenerative Disease Brains: Identification of Reactive Microglia with an Antibody to Variant of CD105/Endoglin

doi: 10.3390/cells8070766

Figure Lengend Snippet: Features of CD105/MAB1097-positive microglia in substantia nigra. ( a ) Control (non-PD case). (panel A) Association of CD105-positive microglia (purple arrows) and IBA-1 positive microglia (brown arrows) with substantia nigra dopaminergic neuromelanin-containing neurons. (panel B) Higher magnification showing interaction of CD105-positive microglia (purple) with degenerating neuromelanin containing dopaminergic neurons. The area imaged in B) is indicated by boxed area in A). Scale bars represent 50 μm. ( b ) ILBD case). (panel A) Association of CD105-positive microglia (purple arrows) and IBA-1-positive microglia (brown arrows) with substantia nigra dopaminergic neuromelanin-containing neurons showing greater numbers of CD105/RnD positive microglia. (panel B) Higher magnification showing greater interaction of CD105-positive microglia (purple arrows) with degenerating neuromelanin-containing dopaminergic neurons and free neuromelanin. Examples of some neuromelanin structures indicated by brown arrowheads. The area of image in B) is indicated by boxed area on A). Scale bars represent 50 υm. ( c ) PD cases. Sparse numbers of CD105-positive microglia (purple arrows) associated with surviving dopaminergic neurons in two separate PD cases panel A and B. The inset in panel B represents the boxed area at higher magnification. Scale bars represent 50 μm).

Article Snippet: CD105 antibodies from R&D Systems (Minneapolis, MN, USA) (MAB1097) or Abcam (Cambridge, MA, USA.) (AB221675) were mixed for 18 hours with recombinant CD105 protein (1097-EN, R&D Systems), which consists of amino acids 26–586 of human CD105 expressed in mouse myeloma cells.

Techniques: Control

Journal: Cells

Article Title: Microglial Phenotyping in Neurodegenerative Disease Brains: Identification of Reactive Microglia with an Antibody to Variant of CD105/Endoglin

doi: 10.3390/cells8070766

Figure Lengend Snippet: CD105/MAB1097-positive microglia associated with pathological structures. ( a ) panels A–B). Strongly CD105-positive microglia were mainly associated with Aβ plaques (panels A, B) but not with phospho-tau positive neurofibrillary tangles (C). CD105/RnD staining observed in p-tau positive neuritic plaque. Scale bars represent 50 μm. ( b ) Confocal colocalization of CD105 and Aβ. Colocalization of CD105/RnD (green) and Aβ (red) and merged image (yellow) demonstrated by confocal microscopy in AD case. The area delineated on Merge image encompasses a microglia with intracellular Aβ (yellow structures). Images are at the same magnification; scale bars represent 20 μm. ( c ) CD105-positive microglia containing neuromelanin. Panels A–D) Strongly positive CD105 positive structures (purple arrows) associated with neuromelanin particles (arrowheads) in the substantia nigra. Panel A, Control case; Panel B, ILBD case; and panel C and D, PD cases. Images are at the same magnification; scale bars represent 50 μm.

Article Snippet: CD105 antibodies from R&D Systems (Minneapolis, MN, USA) (MAB1097) or Abcam (Cambridge, MA, USA.) (AB221675) were mixed for 18 hours with recombinant CD105 protein (1097-EN, R&D Systems), which consists of amino acids 26–586 of human CD105 expressed in mouse myeloma cells.

Techniques: Staining, Confocal Microscopy, Control

Journal: Cells

Article Title: Microglial Phenotyping in Neurodegenerative Disease Brains: Identification of Reactive Microglia with an Antibody to Variant of CD105/Endoglin

doi: 10.3390/cells8070766

Figure Lengend Snippet: Levels of CD105–70 kDa polypeptide in in vitro-stimulated human brain microglia. Top panel: Bar chart showing mean levels of CD105 70 kDa polypeptide in stimulated human microglia. Microglia were stimulated with Aβ(42)(2 μM), poly IC (pIC) (25 μg/mL) or combination of both (pIC-Aβ) in triplicate for 24 hours and analyzed by Western blotting. Significant increase in 70 kDa in pIC-stimulated but not Aβ-stimulated microglia. Results normalized against values for β-actin. This is representative of 3-independent experiments with different microglia isolates. *** p < 0.001; ** p < 0.01. Lower panel: Western blot showing bands in treated microglia (MG). The same 70 kDa band was detected in sample of brain endothelial cells (BEC) isolated from same case. Blots were reprobed with antibody to β actin for normalization purposes.

Article Snippet: CD105 antibodies from R&D Systems (Minneapolis, MN, USA) (MAB1097) or Abcam (Cambridge, MA, USA.) (AB221675) were mixed for 18 hours with recombinant CD105 protein (1097-EN, R&D Systems), which consists of amino acids 26–586 of human CD105 expressed in mouse myeloma cells.

Techniques: In Vitro, Western Blot, Isolation

Journal: Communications Biology

Article Title: Thrombin cleaves membrane-bound endoglin potentially contributing to the heterogeneity of circulating endoglin in preeclampsia

doi: 10.1038/s42003-025-07751-3

Figure Lengend Snippet: The levels of sEng in ( a ) plasma and in ( b ) serum were quantified using an ELISA kit assay in a cohort of 60 patients. This cohort comprised 20 non-pregnant women controls, 20 pregnant women controls, and 20 cases diagnosed with preeclampsia. c Plasma and ( d ) serum samples from preeclampsia patients diluted to ratios of 1:5, 1:10, or 1:20 and reduced with DDT, were subjected to analysis through SDS-PAGE and Western blot with Endoglin/CD105 polyclonal rabbit antibody (ProteinTech). rEng at 100 ng/mL was used as control. The data is presented as mean ± S.D. Statistical significance was determined at * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. NS indicates a non-specific band.

Article Snippet: Recombinant mouse Endoglin/CD105 Fc Chimera Protein, encompassing the extracellular region (Glu27-Gly581), was purchased from R&D Systems (1320-EN/CF). rEng (20 µg/mL) was diluted in Tris-buffered saline polyethylene glycol 1x buffer (TBS-PEG) and treated with human α-thrombin (Thr-H) (Cryopep, 9-HCT-0020-1) or human β-thrombin (Thr-C) (Cambridge ProteinWorks, #10108).

Techniques: Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot, Control

Journal: Communications Biology

Article Title: Thrombin cleaves membrane-bound endoglin potentially contributing to the heterogeneity of circulating endoglin in preeclampsia

doi: 10.1038/s42003-025-07751-3

Figure Lengend Snippet: rEng at a concentration of 20 µg/mL was treated at 37 °C a with increasing concentrations of Thr-H : 0, 0.01, 0.05, 0.1, 0.5, or 1 µM for 1 h. Following treatment, the samples were resolved using SDS-PAGE and subsequently analyzed by Western blotting with Endoglin/CD105 polyclonal rabbit antibody (ProteinTech). b The percentage of all cleaved bands obtained in a by the action of Thr-H on Eng was quantified relatively to the total rEng (70 kDa). c Percentage of the 70 kDa band reduction, specific to the total rEng band from a . d The same procedure described in a was repeated (Eng 20 µg/mL, treatment at 37 °C), but using a fixed concentration of 1 µM of Thr-H for varying durations: 0, 1, 5, 15, 30 or 60 min. e Graph of percentage of all cleaved bands resulting from the action of Thr-H on rEng at various time intervals. f Percentage of the 70 kDa band reduction, specific to the total rEng band from d . left. In a and d the band above 40 kDa (of approximately 50 kDa) was not reproducibly observed in the repeated experiments and was not further studied. The data is presented as mean ± S.D. from three conducted experiments. All images analysis were performed using Image Studio™ Lite software. The dashed line in panel d indicates a cut made in the gel where a blank column had been.

Article Snippet: Recombinant mouse Endoglin/CD105 Fc Chimera Protein, encompassing the extracellular region (Glu27-Gly581), was purchased from R&D Systems (1320-EN/CF). rEng (20 µg/mL) was diluted in Tris-buffered saline polyethylene glycol 1x buffer (TBS-PEG) and treated with human α-thrombin (Thr-H) (Cryopep, 9-HCT-0020-1) or human β-thrombin (Thr-C) (Cambridge ProteinWorks, #10108).

Techniques: Concentration Assay, SDS Page, Western Blot, Software

Journal: Communications Biology

Article Title: Thrombin cleaves membrane-bound endoglin potentially contributing to the heterogeneity of circulating endoglin in preeclampsia

doi: 10.1038/s42003-025-07751-3

Figure Lengend Snippet: a Immunofluorescence staining of Endoglin was conducted using the Endoglin/CD105 P4A4-Alexa 488 antibody (green) and vectashield mounting medium with DAPI (blue) after treating ECFC with increasing concentrations of Thr-H (0.01, 0.1 and 1 µM). Observations were carried out using confocal microscopy at 20x magnification. Phase photos at 10x magnification were taken for each condition to confirm the presence of the cell monolayer. b Endoglin fluorescence intensity following ECFC treatment with increasing concentrations of Thr-H (0.01, 0.1 and 1 µM) was quantified using ImageJ software ( n = 3). c Plots showing the quantification of Eng fluorescence intensity in ECFC following treatment with 1 µM of Thr-H, alongside the number of nuclei relative to the surface. d Immunofluorescence staining of Eng using the Endoglin/CD105 P4A4-Alexa 488 antibody in different cell types, including HUVECs and MSCs, mounted with Vectashield containing DAPI. HUVECs were additionally stained with VE-Cadherin (VE-CAD) using a Texas Red-conjugated antibody, and MSCs were stained with Phalloidin-Alexa 555 to assess cell integrity after treatment with Thr. e Plots showing the quantification of Eng fluorescence intensity in HUVEC and MSC following treatment with 1 µM of Thr-H, alongside the number of nuclei relative to the surface. f ELISA kit assay results for sEng in supernatants of ECFC, HUVEC and MSC treated or not with 1 µM of Thr-H. The data is presented as mean ± S.D. for a minimum of n = 4. Statistical significance was considered at * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Recombinant mouse Endoglin/CD105 Fc Chimera Protein, encompassing the extracellular region (Glu27-Gly581), was purchased from R&D Systems (1320-EN/CF). rEng (20 µg/mL) was diluted in Tris-buffered saline polyethylene glycol 1x buffer (TBS-PEG) and treated with human α-thrombin (Thr-H) (Cryopep, 9-HCT-0020-1) or human β-thrombin (Thr-C) (Cambridge ProteinWorks, #10108).

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Fluorescence, Software, Enzyme-linked Immunosorbent Assay

Journal: Molecular Neurodegeneration

Article Title: Endothelium-specific endoglin triggers astrocyte reactivity via extracellular vesicles in a mouse model of Alzheimer’s disease

doi: 10.1186/s13024-025-00875-4

Figure Lengend Snippet: CEEVs specific endoglin (ENG) is a potential communicator between BMECs and astrocytes in AD. a Schematic and timeline of the transplantation treatment by PKH26-labeled cerebrovascular endothelial extracellular vesicles (CEEVs). b , c The bio-distribution of CEEVs-PKH26 in vivo at 24 h ( b ) and in body tissues 48 h after injection ( n = 3 mice per group) ( c ). d-g Representative images ( d ), the quantification of PKH26 fluorescence intensity in hippocampal dentate gyrus ( n = 3 mice per group) ( e ), the percentage of PKH26 distributed in GFAP staining ( n = 3 mice per group) ( f ), and the quantification of GFAP per astrocyte cell with or without CEEVs from 3 mice per group ( g ). h Differential-gene expression analysis of HCMEC/D3 stimulated by Ang II. Dotted lines indicate HCMEC-DEGs cut-offs for |log2(fold change)|>1.5 and -log10 ( P -value) of 1.3, corresponding to P -value < 0.05, 3 samples per group. i The number of specific genes of HCMEC-DEGs in endothelial cells compared with other cell types in brain (microglia, neuron, astrocyte and oligodendrocyte). j Venn diagrams showing the identification of ENG by intersecting the DEGs of injured BMECs from ( h ) with the differential proteome in the CSF of AD patients and the proteome of endothelial cell EVs from the datasets. k-m A cohort study of AD patients ( n = 18) and non-demented control ( n = 15). ENG protein levels were up-regulated in the serum of AD patients measured by ELISA ( k ). Scatter plot of Montreal Cognitive Assessment (MoCA) versus ENG levels was shown and the data were analyzed with a linear regression method ( l ) and ROC curve analysis of ENG ( m ). Data are shown as means ± SEM, and the P -value was reported on the graph highlighted comparison by unpaired two-sided t -test ( c , e , f , k ) and one-way ANOVA with post-hoc Tukey adjustment ( g ). ENG: Endoglin, ECs: endothelial cells, DEGs: differential genes, NC: Non-demented control

Article Snippet: Mice primary astrocytes were treated with recombinant human ENG protein (SinoBiological, #10149-H02H, China) at 50 ng/mL or SB431542 (MedChemExpress, #HY-10431, USA) at 10 μM for 24 h for qRT-PCR or Western blotting.

Techniques: Transplantation Assay, Labeling, In Vivo, Injection, Fluorescence, Staining, Gene Expression, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Molecular Neurodegeneration

Article Title: Endothelium-specific endoglin triggers astrocyte reactivity via extracellular vesicles in a mouse model of Alzheimer’s disease

doi: 10.1186/s13024-025-00875-4

Figure Lengend Snippet: Injured BMECs deliver ENG protein to astrocytes through CEEVs. a , b Representative images ( a ) and the quantification ( b ) of ENG on per astrocyte in WT and APP/PS1 mice ( n = 5 mice per group). c ENG protein levels in HCMEC/D3 conditioned-medium incubating with or without astrocytes ( n = 6 biologically independent experiments). d , e Immunostaining ( d ) and the quantification ( e ) of ENG expression in HCMEC/D3 treated with Ang II ( n = 6 biologically independent experiments). f ENG protein levels in HCMEC/D3 medium treated by Ang II ( n = 6 biologically independent experiments). g Immunoblotting of distribution of ENG in HCMEC/D3 culture medium. h Protein content of ENG in the CEEVs from Ang II-treated HCMEC/D3 vs. normal control ( n = 3 biologically independent experiments). i , j Representative images ( i ) and the quantification ( j ) of ENG spots on per astrocyte cell in AST & HC Co-culture system treated by GW4869 to HCMEC/D3 or Amiloride to astrocytes from 3 biologically independent experiments. Data are shown as means ± SEM, and the P -value was reported on the graph highlighted comparison by unpaired two-sided t -test ( b , e ), two-way ANOVA with post-hoc Tukey adjustment ( c ), one-way ANOVA with post-hoc Tukey adjustment ( f ) and Kruskal-Wallis with Dunn’s multiple comparisons test ( j ). AST: astrocytes, HC: HCMEC/D3

Article Snippet: Mice primary astrocytes were treated with recombinant human ENG protein (SinoBiological, #10149-H02H, China) at 50 ng/mL or SB431542 (MedChemExpress, #HY-10431, USA) at 10 μM for 24 h for qRT-PCR or Western blotting.

Techniques: Immunostaining, Expressing, Western Blot, Control, Co-Culture Assay, Comparison

Journal: Molecular Neurodegeneration

Article Title: Endothelium-specific endoglin triggers astrocyte reactivity via extracellular vesicles in a mouse model of Alzheimer’s disease

doi: 10.1186/s13024-025-00875-4

Figure Lengend Snippet: CEEVs switch astrocyte reactivity through ENG. a Immunoblotting and the quantification of GFAP in astrocytes treated by ENG recombinant protein ( n = 3 biologically independent experiments). b Immunoblotting of GFAP in astrocytes treated by ENG-overexpressed CEEVs ( n = 3/8 biologically independent experiments). c , d Knockdown of ENG in HCMEC/D3 and immunoblotting of ENG and GFAP in astrocytes treated by ENG-knockdown CEEVs ( n = 3 biologically independent experiments). e-g Representative images ( e ) and the quantification of GFAP in per astrocyte ( f ) and PKH26 fluorescence intensity ( g ) in the hippocampus of CEEVs-treated C57BL/6J mice administrated with or without Carotuximab from 5 mice per group. Data are shown as means ± SEM, and the P -value was reported on the graph highlighted comparison by unpaired two-sided t -test

Article Snippet: Mice primary astrocytes were treated with recombinant human ENG protein (SinoBiological, #10149-H02H, China) at 50 ng/mL or SB431542 (MedChemExpress, #HY-10431, USA) at 10 μM for 24 h for qRT-PCR or Western blotting.

Techniques: Western Blot, Recombinant, Knockdown, Fluorescence, Comparison

Journal: Molecular Neurodegeneration

Article Title: Endothelium-specific endoglin triggers astrocyte reactivity via extracellular vesicles in a mouse model of Alzheimer’s disease

doi: 10.1186/s13024-025-00875-4

Figure Lengend Snippet: ENG regulates reactive astrocytes via TGFBRI/Smad3 pathway. a Immunoprecipitation and immunoblot analysis of ENG and TGFBRI in astrocytes of AST & HC Co-culture system. b Representative images and co-localization analysis of ENG and TGFBRI on astrocytes of AST & HC co-culture system. c Immunoblotting analysis and quantification of Smad3 and phospho-Smad3 (Ser423/425) in primary astrocytes treated with ENG recombinant protein ( n = 3 biologically independent experiments). d The levels of IL-6, IL-3 and VEGF in the medium of ENG-treated astrocytes ( n = 6 biologically independent experiments). e mRNA levels of GFAP and VIM in astrocytes treated by ENG ( n = 6 biologically independent experiments). f Relative mRNA levels of VIM in astrocytes treated by ENG or SB431542 ( n = 5 biologically independent experiments). Data are shown as means ± SEM, and the P -value was reported on the graph highlighted comparison by unpaired two-sided t -test ( c-e ) or one-way ANOVA with post-hoc Tukey adjustment ( f )

Article Snippet: Mice primary astrocytes were treated with recombinant human ENG protein (SinoBiological, #10149-H02H, China) at 50 ng/mL or SB431542 (MedChemExpress, #HY-10431, USA) at 10 μM for 24 h for qRT-PCR or Western blotting.

Techniques: Immunoprecipitation, Western Blot, Co-Culture Assay, Recombinant, Comparison

Journal: Molecular Neurodegeneration

Article Title: Endothelium-specific endoglin triggers astrocyte reactivity via extracellular vesicles in a mouse model of Alzheimer’s disease

doi: 10.1186/s13024-025-00875-4

Figure Lengend Snippet: ENG deficiency inhibits astrocyte reactivity and neuroinflammation in APP/PS1. a Schematic of APP/PS1 experimental procedure of AAV-shENG interference. b Western blotting to identify ENG expression in the hippocampus of AAV-shENG injected APP/PS1 mice ( n = 3 mice per group). c-e Representative images ( c ) and quantification of Smad3 ( d ) and GFAP ( e ) in the hippocampal dentate gyrus of APP/PS1-shCon and APP/PS1-shENG mice ( n = 3 mice per group). f mRNA levels of Serpina3N , VIM and Ctsb in the hippocampus of APP/PS1-shCon and APP/PS1-shENG mice ( n = 6 mice per group). g , h Representative images and quantification of VIM ( g ) and Aβ plaque ( h ) in the hippocampal dentate gyrus of APP/PS1-shCon and APP/PS1-shENG mice ( n = 3 mice per group). i Sholl analysis of astrocytes from the VIM-stained images. Interval of concentric circles is 3 μm. j Average number of intersections in Sholl analysis with respect to the distance from the center of each VIM-stained cell. k , l The sum number of intersects ( k ) and the ending radius ( l ) by Sholl analysis of the stained VIM signal of each VIM-stained cell from 3 mice per group. m Relative mRNA levels of IL-6 , IL-3 and VEGF in the hippocampus of APP/PS1-shCon and APP/PS1-shENG mice ( n = 6 mice per group). Data are shown as means ± SEM, and the P -value was reported on the graph highlighted comparison by unpaired two-tailed t -test

Article Snippet: Mice primary astrocytes were treated with recombinant human ENG protein (SinoBiological, #10149-H02H, China) at 50 ng/mL or SB431542 (MedChemExpress, #HY-10431, USA) at 10 μM for 24 h for qRT-PCR or Western blotting.

Techniques: Western Blot, Expressing, Injection, Staining, Comparison, Two Tailed Test