Fig. 2.
ANXA1 expression modulates in vitro paracellular permeability. (A and B) Paracellular permeability coefficients (A) and transendothelial resistance (TEER; B) measured in WT hCMEC/D3 human brain microvascular endothelial cells or stable hCMEC/D3 clones transfected with a pRc/CMV plasmid alone (empty) or containing an antisense ANXA1 sequence (AS1, AS4, AS100B) or full-length ANXA1 (FL5, FL11); data are representative of at least three independent experiments, performed in triplicate, expressed as mean ± SEM (*P < 0.05 vs. WT clones). (C) Paracellular permeability of WT hCMEC/D3 monolayers following exposure to a neutralizing anti-ANXA1 antibody at 20 µg/mL for 3 h, compared with exposure to an irrelevant mouse IgG isotype control (30 µg/mL); data are representative of at least three independent experiments, performed in triplicate, expressed as mean ± SEM (*P < 0.05). (D) EM analysis of Immunogold labeling for ANXA1 in WT hCMEC/D3 cells grown as a polarized monolayer on a transwell polycarbonate filter reveals a striking localization of the protein at points of cell-cell contact. Upper: Transverse image through the cell monolayer. Lower: Vertical images of cell contacts. (Scale bar: 200 nm.) (E) Confocal microscopic analysis of VE-cadherin and occludin in polarized monolayers of WT, FL11, and AS4 hCMEC/D3 clones reveals marked disturbances in both proteins in AS clones (typical example from n = 3 independent preparations). (Scale bar: 10 µm.)