Article Title: A non-canonical Notch complex regulates adherens junctions and vascular barrier function
Figure Lengend Snippet: Notch1 regulates shear stress-induced vascular barrier function a , Organotypic microfluidic devices of human engineered microvessels (hEMVs) consisting of human ECs (red) in physiologic ECM (green), enabling vessel perfusion at a defined luminal shear stress (inlet and outlet in blue). Inset: 3D reconstruction of hEMVs (red - 70kDa dextran, white – VE-cadherin, green – collagen I). b , Real-time assessment of vascular barrier function in hEMVs cultured statically or under flow (heatmap of fluorescent intensity of 70 kDa dextran, imaging plane indicated by dashed line in a ). c , Quantification of the diffusive permeability (P D ) of 70 kDa dextran across EC barrier as a function of endothelial wall shear stress. d , Relative gene expression for ECs cultured statically or under flow was quantified with qPCR, and Notch target genes regulated by flow are indicated (each column representative of an independent experiment, full gene panel in Extended Data Fig. 2 ). e , ICD cleavage in static and flow EC lysates as measured by western blot with an antibody specific to cleaved ICD (N1 V1754). f , P D measured in hEMVs under static or flow conditions in the presence of Notch inhibitor (DAPT) or rDll4-coated collagen (rDll4). g , Fluorescent micrographs of hEMVs immunostained for VE-cadherin (magenta) and labeled with phalloidin (actin - green) and DAPI (nucleus – blue). h , Quantification of junctional area measured from VE-cadherin immunostained micrographs. i , P D for hEMVs cultured statically, under flow, or in the presence of rDll4-coated collagen with ECs expressing dnMAML, GFP infection control, or with N1-KO, Dll4-KO, or scramble control ECs. j , Fluorescent micrographs of actin and VE-cadherin for hEMVs under flow with ECs expressing dnMAML or with N1-KO ECs. k , Quantification of junctional area measured from VE-cadherin immunostained micrographs (micrographs of GFP and scramble controls in Extended Data Fig. 3 ). For ( a - k ), n≥3 independent hEMVs, mean ± s.e.m. l , Fluorescence intensity heatmaps of Evan’s blue (EB) dye in the mouse dermal vasculature 30min and 60min after IV co-injection of EB and DAPT or DMSO vehicle control. m , Quantification of P D for EB diffusion into the dermal interstitial space (n = 15 vessels across 3 mice/condition). n , Color image of lungs harvested from mice sacrificed 30 min after IV injection of EB. o , Vascular permeability was quantified by eluting and measuring the concentration of EB in lungs relative to the blood EB concentration, (n=4 age, sex-matched littermates, color coded, two-way ANOVA). p , Whole-mount lung vasculature immunostains showing leaked EB and DAPI. *p
Article Snippet: The soluble supernatants were precleared with protein G sepharose beads and then incubated with protein G sepharose beads with VE-cadherin or Trio antibody (Santa Cruz, 1 mg)for 3 hours at 4C, washed three times with lysis buffer and then processed in SDS–PAGE sample buffer.
Techniques: Cell Culture, Flow Cytometry, Imaging, Permeability, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Labeling, Infection, Fluorescence, Injection, Diffusion-based Assay, Mouse Assay, IV Injection, Concentration Assay