Journal: Molecular Genetics & Genomic Medicine

Article Title: Precise CCM1 gene correction and inactivation in patient‐derived endothelial cells: Modeling Knudson's two‐hit hypothesis in vitro

doi: 10.1002/mgg3.755

Figure Lengend Snippet: Generation and characterization of patient‐derived BOECs. (a) Pedigree of the CCM index case (III:2; arrow). (b) Sequence of the heterozygous frameshift variant c.2012delA in the CCM1 gene of III:2. (c) Schematic domain structure for CCM1 and localization of the pathogenic c.2012delA; p.(Asn671Thrfs*36) mutation in its FERM domain. ANK = ankyrin repeat domain; FERM = band Four.1 Ezrin Radixin Moesin; NPxY/F = Asn‐Pro‐X‐Tyr/Phe motif. (d) Brightfield and (e) spheroid sprouting of patient‐derived BOECs. Strong expression of CD31/PECAM‐1 (green) and very few cells expressing SM22α (red, *)(f, g) as well as immunopositivity for CD146 (green, h, i) and CD34 (green, j, k) in BOECs established from a healthy donor (f, h, j) and the index patient III:2 (g, i, k) confirmed their endothelial phenotype. Scale bars indicate 100 µm (f, g) or 400 µm (d, h‐k)

Article Snippet: The following antibodies were used: monoclonal mouse anti‐human CD31 (BBA7, R&D Systems, Minneapolis, MN), polyclonal rabbit anti‐SM22α (ab14106, Abcam, Cambridge, UK), monoclonal mouse anti‐human CD146 (MAB932, R&D Systems), polyclonal rabbit anti‐human CD34 (HPA036723, Sigma‐Aldrich, St. Louis, MO), polyclonal rabbit anti‐KLF4 (PA5‐27441, Thermo Fisher Scientific), secondary goat anti‐mouse IgG antibody, Alexa Fluor 488 (A‐11029, Thermo Fisher Scientific), and goat anti‐rabbit IgG antibody, Alexa Fluor 555 (A‐21429, Thermo Fisher Scientific).

Techniques: Derivative Assay, Sequencing, Variant Assay, Mutagenesis, Expressing

Journal: Biomaterials

Article Title: Cell-based approach for 3D reconstruction of lymphatic capillaries in vitro reveals distinct functions of HGF and VEGF-C in lymphangiogenesis.

doi: 10.1016/j.biomaterials.2015.11.027

Figure Lengend Snippet: Fig. 1. Morphological and molecular characteristics of a human 3D reconstructed lymphatic microvascular network in vitro. (A) Schematic outline of the method for in vitro reconstruction of the lymphatic microvascular network within a connective tissue substitute. Image of the construct at the end of experiment is shown on the right. (B, C) Lymphatic vascular network visualized by immunofluorescent staining for CD31 (red), imaged by confocal microscopy (B), or computationally reconstructed using Imaris software (C). (D) Side view of the 3D network shown in C, at higher magnification. Note lymphatic capillary lumens (white arrows), blind-ends (yellow arrows), and sprouts (green arrows). Inter- connected networks can be seen in two planes, as indicated with the dotted line. (E) Immunostaining for CD31 and Imaris reconstruction showing numerous filopodia protruding from a lymphatic capillary in vitro. (F, G) Double-immunofluorescent staining for podoplanin (F) and CD31 (G) on the whole reconstructed tissue sample. (H) Immunohistochemical staining for Ki67 (brown) in a tissue cross-section. Cell nuclei are shown in blue. Note Ki67þ LECs in the two vessels shown. (I, J) Immunohistochemistry on serial tissue sections for CD31 (I) and Prox-1 (J). (KeN) TEM images showing typical ultra-structural features of lymphatic capillaries: overlapping endothelial cells (K, M, N), button-like adherens junctions (green arrows, K, M, N), discontinuous basement membrane (red arrow in L), anchoring filaments (red arrow in N), single layer of LECs, and lack of mural cell coverage (L, N). Scale bars, 5 mm (A), 500 mm (B, C), 100 mm (D, F, G), 50 mm (H, I, J), 10 mm (E), and 500 nm (KeN). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Whole-mounts and frozen sections were stained with sheep anti-human CD31 (1:100, R&D systems AF806), anti-human podoplanin (1:50, Angiobio 11-003), and corresponding secondary antibodies labeled with Alexa-555 or Alexa-633 (Molecular Probes, Eugene, Oregon, USA).

Techniques: In Vitro, Construct, Staining, Confocal Microscopy, Software, Immunostaining, Immunohistochemical staining, Immunohistochemistry, Membrane

Journal: Biomaterials

Article Title: Cell-based approach for 3D reconstruction of lymphatic capillaries in vitro reveals distinct functions of HGF and VEGF-C in lymphangiogenesis.

doi: 10.1016/j.biomaterials.2015.11.027

Figure Lengend Snippet: Fig. 3. Effects of HGF/c-Met and VEGF-C/VEGFR-3 inhibition on 3D in vitro lymphangiogenesis. (A, B) Formation of lymphatic microvascular network in the presence of an anti- VEGFR-3 blocking antibody (2.5 mg/ml), c-Met inhibitor SU11274 (1 mM), or both. Lymphatic vasculature was visualized by immunostaining of the whole construct with a CD31 antibody and reconstructed in 3D with the Imaris software. B is a higher magnification of an area shown in A. (C) Cross-sections of the LEC-fibroblast constructs treated with an anti-VEGFR-3 antibody, c-Met inhibitor or both as indicated, and immunostained for the lymphatic marker podoplanin. (DeG) Quantitative analyses of the lymphatic network volume (D), number of vessels (E), vessel size distribution (F) and connectivity (G) upon treatments as indicated. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 500 mm (A), 50 mm (B), 100 mm (C).

Article Snippet: Whole-mounts and frozen sections were stained with sheep anti-human CD31 (1:100, R&D systems AF806), anti-human podoplanin (1:50, Angiobio 11-003), and corresponding secondary antibodies labeled with Alexa-555 or Alexa-633 (Molecular Probes, Eugene, Oregon, USA).

Techniques: Inhibition, In Vitro, Blocking Assay, Immunostaining, Construct, Software, Marker

Journal: Cell transplantation

Article Title: Human Placenta-Derived Mesenchymal Stem Cells Combined With Artificial Dermal Scaffold Enhance Wound Healing in a Tendon-Exposed Wound of a Rabbit Model.

doi: 10.1177/09636897241228922

Figure Lengend Snippet: Figure 8. Expression of CD31 and human nuclei. (A) Paraffin sections of skin lesions from autologous skin transplantation for 1 week were obtained, and the endothelial cells CD31 and human nuclei were stained by immunohistochemistry. Scale bars: 100 μm (upper row), 20 μm (lower row). (B) Quantification of CD31-positive expression. hPMSCs: human placenta-derived mesenchymal stem cells.

Article Snippet: After de-paraffinizing with xylene and rehydrating the sections (descending gradient of ethanol), primary antibodies against CD31 (1:1,000, OriGene, USA) or human nuclear antibody (1:500, Millipore, USA) were added at 4°C overnight, followed by incubation with horseradish peroxidaseconjugated affinipure goat anti-mouse immunoglobulin G (IgG) for 1 h at room temperature, followed by colorimetric detection using DAB (3,3′-diaminobenzidine) substrate, according to the manufacturer’s protocol20.

Techniques: Expressing, Transplantation Assay, Staining, Immunohistochemistry, Derivative Assay

Journal: Breast cancer research : BCR

Article Title: Dissecting the tumor microenvironment in primary breast angiosarcoma: insights from single-cell RNA sequencing.

doi: 10.1186/s13058-025-02022-9

Figure Lengend Snippet: Fig. 1 Imaging and pathological analysis of a breast angiosarcoma patient. MRI reveals two masses in the central and lower outer quadrants of the right breast (A). CT scan shows a large mass in the left breast and multiple masses in the right breast (B). H&E staining at 40× magnification shows primary angiosarcoma of the breast, with tumor tissue exhibiting diverse morphologies. Interconnecting slit-like structures are visible, with well-formed anastomosing blood vessels at the periphery. The central region displays a higher tumor cell density and dilated lumina (C). H&E staining at 200× magnification reveals dense areas where tumor cells grow in sheets. The cells exhibit spindle-shaped and epithelioid morphologies, with visible mitotic figures. Blood lakes are present within variably shaped and dilated blood vessels (D). Immunohistochemical staining for CD31, CD34, Factor VIII, and FLI-1 shows variable expression across these markers (E-H)

Article Snippet: Immunostaining was conducted using a Dako Autostainer (Dako Corporation, Carpinteria, CA), and the following antibodies were employed: anti-CD31 rabbit polyclonal antibody (ZA-0658, Clone EP78, Beijing Golden Bridge Biotechnology), anti-CD34 monoclonal antibody (Kit-0004, Clone QBEnd/10, Fuzhou Maixin Biotechnology), anti-Factor VIII monoclonal antibody (ZM-0022, Clone OTI9 F3, Beijing Golden Bridge Biotechnology), and anti-FLI-1 monoclonal antibody (ZM-0108, Clone G146-22, Beijing Golden Bridge Biotechnology).

Techniques: Imaging, Computed Tomography, Staining, Immunohistochemical staining, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Conditions Mimicking the Cancer Microenvironment Modulate the Functional Outcome of Human Chorionic Villus Mesenchymal Stem/Stromal Cells in vitro

doi: 10.3389/fcell.2021.650125

Figure Lengend Snippet: Effect of CM-MDA231 on CV-MSCs expression of Adhesion molecules. Flow cytometry analysis of the adhesion molecules performed on preconditioned, in-treatment, and untreated CV-MSCs showed that there was significant increase in the expression levels for VCAM, ICAM1 and PECAM, after preconditioning them for 72 h (A–C) , but no changes in the expression levels were observed in cells preconditioned for 24 h, in-treatment and untreated controls. No significant changes in expression levels was observed for E-Cadherin (D) between the preconditioned, in-treatment and untreated controls (Panel 1). The data obtained by FACS from five independent experiments was quantified. The average is presented as bar diagrams in panel 2. VCAM is represented as (A) , ICAM1 as (B) , PECAM as (C) and E-Cadherin as (D) in panel 2, respectively. Bars represent standard errors * P ≤ 0.05.

Article Snippet: GAPDH and a panel of fluorescent-labeled antibodies (VCAM (cat#FAB5649P), ICAM (cat#BBA20), PECAM (cat#FAB3567P), E-Cadherin (cat#FAB18381P), Integrin α5 (cat#FAB1864P), Integrin-M (cat#FAB16991P), and EpCAM (cat#FAB9601P), used for flow cytometry were purchased from R&D Systems, MN, United States. p53 (cat#2527), pRb (S807) (cat#8516), pChk2 (T68) (cat#2661) and β-Actin (cat#3700 and cat#8457) antibodies for immunoblotting were purchased from Cell Signaling Technologies, MA, United States.

Techniques: Expressing, Flow Cytometry