Journal: PLoS ONE

Article Title: Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells’ Migration

doi: 10.1371/journal.pone.0154709

Figure Lengend Snippet: Shed membrane vesicles obtained from endothelial cells cultured to confluence (C) or in migrating (M) conditions were assayed for their capability to degrade different ECM components, in particular they were assayed on basal lamina components, Laminin (LM), Type-IV collagen (Coll-IV) and Fibronectin (FN) (a), and on type-I collagen fibrils (b and c). In (a), we see the degradative patterns obtained by treatment for 24 hrs of basal lamina components. Type-I and IV collagens and Laminin were biotinilated and degradation stained by streptavidin conjugated to HPR and developed with ECL staining (Amersham); while Fibronectin (FN) degradation was stained with mAbs against human Fibronectin. In (b) we see the kinetic degradative patterns obtained by treating jellified type-I collagen with vesicles obtained in different cell culture conditions at different times of incubation. In (c) there are two controls; collagen fibrils treated with MMP-2 originated by molecular engineering (MMP-2) and spontaneous degradation of type-I collagen fibrils (Control) at different times. In (a) markers are in kDa; in (b) and (c) the positions of α 1 (I) and α 2 (I) monomers of interstitial type-I collagen are marked.

Article Snippet: Fibronectin peptides were analyzed by immunoblotting using mAb against Fibronectin (Usbiological) and stained with HPR conjugated streptavidin and the ECL system (Amersham).

Techniques: Membrane, Cell Culture, Staining, Incubation, Control

Journal: Free radical biology & medicine

Article Title: Endothelial-Transcytosed Myeloperoxidase Activates Endothelial Nitric Oxide Synthase via a Phospholipase C-Dependent Calcium Signaling Pathway

doi: 10.1016/j.freeradbiomed.2020.12.448

Figure Lengend Snippet: (A) Confluent ECs were incubated with MPO (20 nM) for the indicated times (0-120 min) and cellular levels of eNOS and MPO assessed in cell lysates by Western Blotting. (B, C) Confluent ECs on coverslips were incubated with MPO (20 nM) for 2 h, washed and then fixed with paraformaldehyde and immunostained for MPO (red) or fibronectin (green) by laser confocal fluorescence microscopy. Serial images were taken along the z-plane using the z-stack feature included in the microscopy software. Images were processed using ImageJ, where the apical, intracellular and basolateral planes were determined by referencing the DAPI-stained cell nuclei (blue).

Article Snippet: Fixed cells were probed with a rabbit polyclonal antibody against MPO (1:300; Merck Millipore) or a mouse monoclonal antibody against fibronectin (1:100; Clone E3E, Merck Millipore), followed by the relevant labelled secondary antibody; i.e., cyanine 2-conjugated goat anti-mouse (green) or cyanine 3-conjugated donkey anti-rabbit (red) antibodies.

Techniques: Incubation, Western Blot, Fluorescence, Microscopy, Software, Staining

Journal: Oncotarget

Article Title: Upregulated TRIM29 promotes proliferation and metastasis of nasopharyngeal carcinoma via PTEN/AKT/mTOR signal pathway

doi: 10.18632/oncotarget.7215

Figure Lengend Snippet: A. Western blotting reveals a reduced expression of epithelial makers (E-cadherin and α-catenin) and an increased expression of mesenchymal markers (fibronectin and vimentin) in TRIM29-expressing S-18 and 6-10B cells in comparison with the vector controls. GAPDH was used as a loading control. B. Western blotting assay shows increased levels of E-cadherin and α-catenin, and decreased levels of fibronectin and vimentin in 5-8F-shTRIM29 cells compared with that in shNC treated cells. C. Over-expression of TRIM29 promoted S-18 cell invasion and metastasis in athymic nude mice in vivo . Left, representative images show lungs with metastatic NPC tumors; left (lower), number of metastatic nodules formed in the lung of mice 6 weeks after tail vein injection of S-18-vector and S-18-TRIM29 cells (5 mice per group; p < 0.01; independent Student's t- test); right, Examples of haematoxylin and eosin staining in two lung samples originating from S-18-vector and S-18-TRIM29 cell-injected mice (magnification 400×).

Article Snippet: Rabbit polyclonal antibody against human TRIM29 was purchased from Abcam; mouse monoclonal antibodies against human α-catenin, E-cadherin, fibronectin and vimentin were purchased from BD Biosciences; rabbit monoclonal antibodies against human phospho-AKT (Ser473), AKT (pan), phospho-p70S6K (Thr389), p70S6K, phospho-4E-BP1, 4E-BP1 and PTEN were obtained from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Plasmid Preparation, Over Expression, In Vivo, Injection, Staining

Journal: American Journal of Cancer Research

Article Title: Tannic acid attenuates the formation of cancer stem cells by inhibiting NF-κB-mediated phenotype transition of breast cancer cells

doi:

Figure Lengend Snippet: TA reduces TGFβ-induced EMT and cancer stem cell properties in MCF7 and T47D cells. (A) TGFβ treatment (10 ng/ml) induced morphological changes in the MCF7 and T47D cells, (including a loss of cell contact and elongation), which was ameliorated by treatment with TA (10 µM). (B) TA treatment alleviated the TGFβ-induced decrease and cytoplasmic localization of E-cadherin (red) in the MCF7 cells. Representative photos of cell morphology and immunofluorescent staining are shown, Nuclei are stained with DAPI (blue). Magnification × 200. (C, D) Real-time PCR (C) and western blot analysis (D) (with quantitation bar) showing the TGFβ-induced EMT shown as the decreased expression of the epithelial cell marker, E-cadherin (E-cad), cytokeratin 18 (CK18), and increased expression of the mesenchymal marker, N-cadherin (N-cad), Vimentin and Fibronectin. (E, F) TGFβ treatment enhanced mammosphere formation on days 3 and 7 after the initiation of treatment. TA treatment (10 μM) subsequently reduced both the MFE and average mammosphere size. Representative photos of mammosphere are shown (E). Scale bar = 100 μm. (G) TA treatment ameliorated the TGFβ-induced alteration in CD44high/CD24low expression. β-actin was used as a loading control. The data shown represent the mean ± SEM of four independent experiments. *P < 0.05 vs. control; #P < 0.05 vs. TGFβ.

Article Snippet: The membranes were blocked (30 min, room temperature) in 3% bovine serum albumin (BSA) in PBS-Tween 20 (0.1%, v/v), and then incubated (overnight, 4°C) with primary antibodies against E-cadherin, N-cadherin, Fibronectin (BD Biosciences, San Jose, CA, USA), p-IκBα, IκBα, CD44, pIKK, IKK (Cell Signaling Technology, Danvers, MA, USA), cytokeratin-18 (CK18), Vimentin, CD24, IL-6, ALDH1, p65, LaminB and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Staining, Real-time Polymerase Chain Reaction, Western Blot, Quantitation Assay, Expressing, Marker

Journal: Cancer cell

Article Title: Loss of pVHL is sufficient to cause HIF dysregulation in primary cells but does not promote tumor growth

doi:

Figure Lengend Snippet: A & B: Hematoxylin and eosin staining of Vhl+/- and Vhl-/- tumors. C & D: Basement membrane expression of fibronectin in Vhl+/- endothelial cells (arrows) and reduced fibronectin deposition in endothelial cells of Vhl-/- tumors (arrows). E: CD-34 staining of Vhl+/- microvessels. F: Microvessel density in Vhl-/- tumors appeared to be increased as demonstrated by α-CD-34 staining. G: Quantitation of tumor microvessel density. The microvessel density of Vhl-/- tumors was greater than controls, n=3. Student t-test revealed statistically significant differences between tumor types, p<0.03. Bars represent standard error. Final magnifications are 100X (A and B), 400X (C and D) and 200X (E and F)

Article Snippet: 6μm sections of each sample were incubated overnight with antibodies generated against fibronectin (BD Scientific), cleaved caspase-3 (Calbiochem), Ki-67 (Novacastra), and CD-34 (BD Scientific).

Techniques: Staining, Expressing, Quantitation Assay