Journal: The Journal of Experimental Medicine

Article Title: Piezo1 and G q /G 11 promote endothelial inflammation depending on flow pattern and integrin activation

doi: 10.1084/jem.20180483

Figure Lengend Snippet: Integrins are differentially activated by different flow patterns via Piezo1 and G q /G 11 . (A–C) HUAECs were transfected with scrambled siRNA (control) or siRNA directed against ITGA5 and ITGAV (A) or against Gα q and Gα 11 (Gα q/11 ; B and C) and were exposed to laminar or disturbed flow as described in . Activation of integrin signaling was determined by immunoblotting for phosphorylated focal adhesion kinase (pFAK, Y397; A) or by immunoprecipitation of activated α5 integrin (B and C). P65 activation was determined by anti–phospho-P65 (S536) antibodies (A). Bar diagrams show densitometric evaluation of immunoblots (quantification of three to five independent experiments). (D) Ldlr-KO mice without or with endothelium-specific Gα q /Gα 11 deficiency (Ldlr-KO;EC-Gα q/11 -KO) endothelium-specific Piezo1-deficiency (Ldlr-KO;EC-Piezo1-KO) were fed a high-fat diet for 4 wk ( n = 4–6 mice per genotype). Cross sections of the inner and outer curvatures of aortic arches were immunostained with antibodies against activated α5 integrin (SNAKA51; green), against the endothelial marker CD31 (red) or with DAPI (blue). Bar diagrams show percentage of area stained by anti-activated α5 integrin antibody of total endothelial cell area defined by staining by anti-CD31 antibody (based on analysis of at least five sections each from at least four different animals). Bars, 25 µm. Data represent mean ± SEM; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (two-tailed Student’s t test).

Article Snippet: Cells were then incubated in flow chambers with primary antibody directed against activated integrin α5, SNAKA51 (Novus Biologicals, NBP2-50146), overnight at 4°C (1:100 dilution).

Techniques: Transfection, Control, Activation Assay, Western Blot, Immunoprecipitation, Marker, Staining, Two Tailed Test

Journal: Cells

Article Title: Decidua Parietalis Mesenchymal Stem/Stromal Cells and Their Secretome Diminish the Oncogenic Properties of MDA231 Cells In Vitro

doi: 10.3390/cells10123493

Figure Lengend Snippet: Effect of DPMSCs on MDA231 cell expression of adhesion molecules. Flow cytometry analysis of the adhesion molecules performed on DPMSC-treated MDA231 cells or untreated controls showed a significant decrease in the expression levels for PECAM in IC, SF, and CM settings as compared to the untreated control ( A ). EpCAM/TROP-1 was reduced significantly in MDA231 cells in the SF setting as compared to IC and CM settings, as well to the untreated control ( B ). The expression of integrin alpha 2/CD49e, showed a significant decrease in SF and CM treatment conditions ( C ), whereas ICAM1 was significantly reduced in all the treatment conditions tested ( D ). Bar graphs represent the data obtained from three independent experiments and are shown as mean florescence index (MFI) units pertaining to CD31/PECAM1 ( A ( i )), EpCAM/TROP1 ( B ( i )), integrin alpha 5/CD49e ( C ( i )), and ICAM1 ( D ( i )). Bars represent standard errors. * p < 0.05.

Article Snippet: Fluorescent-labeled antibodies for flow cytometry experiments, including GAPDH (cat#AF5718); ICAM (cat#BBA20); PECAM (cat#FAB3567P); integrin α5 (cat#FAB1864P); and EpCAM (cat#FAB9601P) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Flow Cytometry, Control

Journal: iScience

Article Title: Anti-angiogenic collagen IV-derived peptide target engagement with α v β 3 and α 5 β 1 in ocular neovascularization models

doi: 10.1016/j.isci.2023.106078

Figure Lengend Snippet:

Article Snippet: C overnight with rabbit antibodies directed against α v β 3 (1:100, Abbiotec, Escondido, CA; Cat # 251672), α 5 β 1 (1:100, Novus Biological, Centennial, CO; Cat # nbp2-29788), α 5 (1:200, Millipore Sigma, Burlington, MA; Cat # AB1928), α v (1:200, Millipore Sigma, Burlington, MA; Cat # AB1930) or AXT107 (1:100, Asclepix, Baltimore, MD) in blocking solution.

Techniques: Recombinant, Protease Inhibitor, Electron Microscopy, Plasmid Preparation, Transgenic Assay, Software, Diagnostic Assay, Microinjection

Journal: PLoS ONE

Article Title: S . aureus alpha-toxin monomer binding and heptamer formation in host cell membranes – Do they determine sensitivity of airway epithelial cells toward the toxin?

doi: 10.1371/journal.pone.0233854

Figure Lengend Snippet: A: Representative examples of immune fluorescence assays using epifluorescence microscopy (nuclei counterstained using DAPI) that were performed on 16HBE14o-, S9 or A549 cells grown on coverslips using antibodies against ADAM10, the α5β1 integrin or against caveolin-1. Staining appearing in red represents specific labelling of the respective proteins. Scale bars: 10 μm. B: Semi-quantitative determination of primary and secondary antibody-mediated fluorescence in suspended individual cells by flow cytometry. During flow cytometry, the fluorescence of the antibody-tagged proteins per cell was measured and the respective medians of the detected peaks were used for calculating the means ± S.D. for the biological replicates (n = 4, each). Individual means were tested for significant differences using Student’s t-test or Welch’s t-test: * = p ≤ 0.05, ** = p ≤ 0.01 or *** = p ≤ 0.001.

Article Snippet: Antibodies (Ab) were obtained from these sources: Hla-Ab (S7531) from Sigma (Steinheim, Germany); ADAM10 Ecto (MAB1427-100) from R&D Systems and purchased through antikoerper-online.de (Aachen, Germany); Caveolin-1 (7C8) (sc-53564), normal mouse IgG 2b (sc-3879) from Santa Cruz Biotechnology (Heidelberg, Germany); Integrin α5β1 (M200) (NBP2-52680) from Novus Biologicals and purchased through Bio-Techne (Wiesbaden, Germany); Alexa Fluor ® 594 AffiniPure goat anti-mouse IgG (H+L) (115-585-003) from Jackson ImmunoResearch and purchased through Dianova (Hamburg, Germany); goat anti-rabbit IgG-HRP (7074s) and anti-rabbit IgG (H+L) F(ab') 2 Fragment Alexa Fluor ® 594 Conjugate (8889S) from Cell Signaling (Frankfurt am Main, Germany).

Techniques: Fluorescence, Epifluorescence Microscopy, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Galectin-1 Restricts Vascular Smooth Muscle Cell Motility Via Modulating Adhesion Force and Focal Adhesion Dynamics

doi: 10.1038/s41598-018-29843-3

Figure Lengend Snippet: Characterization of interaction of Gal-1 with α5β1 integrin and fibronectin. (A) SPR analysis of interaction between CSGal-1 and α5β1intergrin. Recombinant mouseα 5β1intergrin protein was immobilized on a CM5 chip and CSGal-1 at indicated concentrations were injected at a flow rate of 30 μl/min. Left panel, sensorgrams generated with serial concentrations of CSGAL-1. Middle panel, the curve showing the specific signals obtained after subtracting the background binding to control chip without α5β1intergrin immobilization. Right panel, sensorgrams of CSGal-1 (1600 nM) pre-incubated without (control) or with 10 mM sucrose or lactose prior to injection. (B) Human plasma fibronectin was immobilized on a CM chip and CSGAL-1 at indicated concentrations were injected at a flow rate of 30 μl/min. Left panel, sensorgrams generated with serial concentrations of CSGAL-1. Middle panel, the curve showing the specific signals obtained after subtracting the background binding to control chip without fibronectin immobilization. Right panel, sensorgrams of CSGal-1 (6400 nM) pre-incubated without (control) or with 5 mM sucrose or lactose prior to injection. (C) WT and Gal-1-KO VSMCs were reseeded on fibronectin-coated dishes for 16 h. Cells were incubated with 1 mM 3,3-dithiobis[sulfosuccinimidylpropionate] in PBS at 4 °C for 30 min, followed by addition of 50 mM Tris-HCl to quench unreacted crosslinker. Cells were then extracted with PBS containing 100 mM lactose, 0.1% SDS and 0.5% Triton X-100. After 3 PBS washes, crosslinked proteins were recovered by incubation with 50 mM dithiothreitol in PBS at 37 °C for 30 min. Both un-crosslinked and crosslinked proteins were subjected to Western blot analysis with specific antibodies targeting respective proteins. Uncropped images of immunoblots are shown in Supplementary Fig. .

Article Snippet: The 96-well polystyrene high bind microplate (#9018; Corning) was incubated with 100 μl of 10 μg/mL recombinant mouse α5β1intergrin protein (7728-A5, R&D systems) in 50 mM Na 2 CO 3 pH 9.6 at 4 °C overnight.

Techniques: Recombinant, Injection, Generated, Binding Assay, Control, Incubation, Clinical Proteomics, Western Blot