Figure 3. Confocal microscopy of endothelial junctions after PAR-1 activation. (A) Representative micrographs of anti–VE-cadherin staining. After transfection with either human FoxM1 siRNA (siRNA) or scRNA, HMVEC-L were plated in 24-well plates with a glass coverslip in each chamber at 100% confluency. At 65 h after transfection, the cells were treated with 4 U/ml thrombin in triplicate, and fixed with 4% paraformaldehyde at the indicated times. The fixed cells were immunostained with anti–VE-cadherin antibody (green) to visualize junctions, and nuclei were counterstained with DAPI (blue). FoxM1-deficient HMVEC-L (siRNA-transfected) exhibited defective reannealing of AJs at 2 and 4 h after thrombin challenge, in contrast to scRNA-transfected controls (scRNA). Arrows indicate failure of reannealing of AJs. The experiment was performed three times with similar data. Bar, 50 µm. (B) Quantification of intercellular AJ gap area. The area of intercellular AJ gaps was quantified using MetaMorph 7.1.0 by manually outlining cells and selecting for gaps. Values are expressed as the percentage of total surface area. Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus scRNA at 120 and 240 min after thrombin challenge.
Figure 3.

Confocal microscopy of endothelial junctions after PAR-1 activation. (A) Representative micrographs of anti–VE-cadherin staining. After transfection with either human FoxM1 siRNA (siRNA) or scRNA, HMVEC-L were plated in 24-well plates with a glass coverslip in each chamber at 100% confluency. At 65 h after transfection, the cells were treated with 4 U/ml thrombin in triplicate, and fixed with 4% paraformaldehyde at the indicated times. The fixed cells were immunostained with anti–VE-cadherin antibody (green) to visualize junctions, and nuclei were counterstained with DAPI (blue). FoxM1-deficient HMVEC-L (siRNA-transfected) exhibited defective reannealing of AJs at 2 and 4 h after thrombin challenge, in contrast to scRNA-transfected controls (scRNA). Arrows indicate failure of reannealing of AJs. The experiment was performed three times with similar data. Bar, 50 µm. (B) Quantification of intercellular AJ gap area. The area of intercellular AJ gaps was quantified using MetaMorph 7.1.0 by manually outlining cells and selecting for gaps. Values are expressed as the percentage of total surface area. Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus scRNA at 120 and 240 min after thrombin challenge.

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