FIG. 2.
cAMP induces AJ formation. (A) HUVECs cultured on a glass base dish were stimulated with 10 μg of PGI2/ml (upper panels) or with 10 μM FSK (lower panels) for 20 min and shown as phase-contrast images. Left and right panels show the cells before and after stimulation, respectively. The arrows indicate the sites of cell-cell contacts induced by PGI2 and FSK. The area boxed by the white broken line is enlarged in the right top of the panels. Bars, 50 μm. (B) Subconfluent HUVECs stimulated with vehicle (Cont), 10-μg/ml PGI2, and 10 μM FSK for 45 min were fixed, stained with anti-VE-cadherin antibody, and visualized with Alexa 488-conjugated secondary antibody through a confocal microscope (BX50WI; Olympus). Note that VE-cadherin (green) was accumulated at the cell-cell contact upon PGI2 and FSK stimulation. Bars, 50 μm. (C) Translocation of VE-cadherin was assessed by Triton X-100 solubility. HUVECs were stimulated with vehicle (top), 10-μg/ml PGI2 (middle), and 10 μM FSK (bottom) for the time indicated at the top and fractionated with cytoskeleton-stabilizing buffer as described in Materials and Methods. The Triton X-100-insoluble fraction was subjected to SDS-PAGE followed by Western blot analysis (WB) with anti-VE-cadherin.