Journal: Nature Communications

Article Title: Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors

doi: 10.1038/ncomms15374

Figure Lengend Snippet: ( a – c ) ECs increase the levels of active β1 integrin in hfRPE basal plasma membrane. ( a , b ) Active β1 integrin (blue) and the TJ protein ZO-1 (red) in hfRPE were assessed by immunofluorescence assays (10 z-stacks from 2 biological replicates, t -test). Top panel, orthogonal view (xz) of a confocal z-stack. Bottom panel, confocal plane (xy) indicated by the arrowhead in the top panel. Bar, 10 μm. ( c ) Example of the relative fluorescence of individual confocal planes from 5 z-stacks is shown from the most apical (left) to the most basal (right) planes. ( d ) Inhibition of hfRPE β1 integrin with a blocking antibody impairs EC-mediated increase in TER. For clarity, only the statistical analyses at day 7 are shown ( n =6, ANOVA), all groups versus VeraVec ECs+anti-β1 integrin). ( e , f ) RhoA/ROCK and Rac1 pathways are involved in the EC-mediated increase in hfRPE TER. For clarity in e , only the statistical analyses at week 2 are shown. In e : *Mock control versus mock+ROCK inhibitor; && VeraVec ECs control versus VeraVec ECs+Rac1 inhibitor ( n =6, ANOVA). In f : ***Mock control versus mock+RhoA activator; &&& VeraVec ECs control versus VeraVec ECs+RhoA activator ( n =3, ANOVA). ( g , h ) Cell surface biotinylation assays showing EC-mediated increase in occludin cell surface localization in hfRPE ( n =4, t -test). ( i , j ) Immunofluorescence assays show a lysyl oxidase activity-dependent enhancement of occludin accumulation (green) along ZO-1-positive TJs (red) in hfRPE when exposed to choroid EC-conditioned media ( n =5, ANOVA). Bar, 20 μm. ( k ) Rac1 inhibition impairs EC-mediated occludin accumulation at hfRPE TJs [10 (mock and VeraVec ECs) or 12 (VeraVec ECs+Rac1 inhibitor) z-stacks from 2 biological replicates, ANOVA). ( l , m ) Increasing substrate stiffness enhances occludin localization at hfRPE TJs (10 z-stacks from 2 biological replicates, ANOVA). Bar, 10 μm. ( n ) Inhibiton of lysyl oxidase acitivity does not impair EC-mediated accumulation of collagen I in hfRPE basement membrane (40 images per condition from 2 biological replicates, ANOVA). Data in c – f , h and j are presented as mean±s.d.

Article Snippet: Where indicated, medium in the bottom chamber of Transwell inserts was supplemented with 8 μg ml −1 control rat IgG1 (clone HRPN, cat. MABF1786) or blocking anti-β1 integrin antibody (clone AIIB2 (ref. ), cat. MABT409) (EMD Millipore).

Techniques: Clinical Proteomics, Membrane, Immunofluorescence, Fluorescence, Inhibition, Blocking Assay, Control, Activity Assay

Journal: The Journal of Cell Biology

Article Title: Reversion of the Malignant Phenotype of Human Breast Cells in Three-Dimensional Culture and In Vivo by Integrin Blocking Antibodies

doi:

Figure Lengend Snippet: Comparison of Various β1-Integrin Antibody Treatments on Colony Size in 3-Dimensional Culture

Article Snippet: Rat monoclonal AIIB2 IgG 1 's were isolated using Affi-Gel Protein A Maps II system (Bio-Rad Labs, Hercules, CA).

Techniques: Comparison, Control, Purification