Journal: Current issues in molecular biology
Article Title: The Vitamin K-Dependent Anticoagulant Factor, Protein S, Regulates Vascular Permeability.
doi: 10.3390/cimb46040205
Figure Lengend Snippet: Figure 1. Human PROS1 increases endothelial monolayer permeability and activates the p38 MAPK and Rho/ROCK pathways. (A) Fold increase in EC monolayer permeability over control (diluent) induced by exposing ECs to either human PROS1 (10 µg/mL, 24 h) or VEGFA (20 ng/mL, 1 h) or a combination of human PROS1 (10 µg/mL, 24 h) followed by VEGFA (20 ng/mL, 1 h). For permeability assay, ECs plated on filter inserts were switched to EBM-2 and exposed to PROS1 and or VEGFA. 40 kDa FITC-dextran (1 mg/mL) was then added to the upper filter chamber for 30 min then 100 µL of the culture media of the lower chamber were removed, fluorescence was read (excitation 488 nm, emission 525 nm), and results are expressed as percentage of control (diluent) in fluorescence. (B) Permeability was assessed as in (A); results are expressed as percentage of control (vehicle) in EC layer permeability induced by exposing cells to either human PROS1 (10 µg/mL, 24 h) or a combination of human PROS1 (10 µg/mL, 24 h) and anti-human PROS1 IgG (10 µg/mL, 24 h) or human PROS1 (10 µg/mL, 24 h) and an irrelevant non-immune IgG (10 µg/mL, 24 h). Data in (A,B) are from 6 and 3 independent experiments, respectively, each performed in triplicate, and are expressed as percentage of control (diluent) ± SEM. **** p < 0.0001, *** p < 0.001, ** p < 0.01, ns indicates not significant. (C,D) Western blot analysis of HSP27 and MLC phosphorylation in cultured ECs. Subconfluent EC cultures were switched to EBM-2 plus PROS1 (10 µg/mL) or diluent for 15, 20, or 60 min. Cells were then lysed, and equivalent amounts of protein were analyzed for phospho-HSP27 or phospho-MLC2; band intensities were quantified and are represented as a percentage of the ratio of, respectively, phosphorylated HSP27 and MLC over actin. Data are from 6 and 4 independent experiments, respectively, each performed in triplicate, and expressed as percentage of control ± SEM. Statistical analyzes were performed using a Mann–Whitney test in order to compare each treatment time to the control ** p < 0.01, * p < 0.05. (E) Subconfluent ECs cultured on fibronectin-coated slides were exposed to either human PROS1 (10 µg/mL) or diluent for 1 h in EBM-2 medium. Cells were fixed, permeabilized and stained with Texas-Red phalloidin and DAPI. Slides were then analyzed by confocal microscopy. Arrows indicate changes in actin staining intensity, and white scale bars represents 50 µm length. (F) Quantification of actin staining intensity by Image v.J2 software. Results are presented as the ratio of the F-actin intensity/cell numbers and are expressed as the mean ± SEM of three independent experiments. Statistical analysis was performed using Prism 4.2 software.
Article Snippet: Cells were then fixed in 4% paraformaldehyde in PBS, permeabilized in 0.1% Triton X-100 in PBS and incubated with anti-VEC monoclonal antibody (BD Biosciences; Franklin Lakes, NJ, USA) (Table 1) followed by sequential incubations with biotin-labelled anti-mouse/anti-rabbit immunoglobulin G (Table 1) and fluorescein isothiocyanate (FITC)-labelled avidin D (Vector Laboratories), the latter was added together with 4 U/mL Texas Red–Phalloidin (Invitrogen; Thermo Fisher; Waltham, MA, USA) for simultaneous staining of F-actin.
Techniques: Permeability, Control, Fluorescence, Western Blot, Phospho-proteomics, Cell Culture, MANN-WHITNEY, Staining, Confocal Microscopy, Software
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