PKA inhibits endothelial cell migration. (A) Number of endothelial cells ± SEM migrating on vitronectin in serum-free culture medium or in culture medium containing PTHrP or isoproterenol, *P < .05. (B) Relative levels of PKA activity ± SEM in endothelial cells treated with culture medium or 20 μg/mL forskolin, *P < .05. (C) Relative levels of activity ± SEM in ECs after transfection with pcDNA 3.1 V5/his-dominant negative PKA (dnPKA) or N1GFP (GFP) with or without treatment with forskolin, *P < .05. (D) Number of endothelial cells ± SEM migrating on vitronectin in the absence (filled bars) or presence of forskolin, *P < .05. (E) Number of endothelial cells ± SEM migrating on vitronectin after transfection with pcDNA 3.1 V5/his-PKA catalytic subunit and N1GFP (PKA Catalytic) or N1GFP (GFP) vectors, *P < .05. (F) Number of endothelial cells ± SEM migrating on vitronectin after transfection with pcDNA 3.1 V5/his-RImut (dnPKA) or N1GFP treated with culture medium or forskolin, *P < .05. (G) Freeze frame photographs at 200× and 400× from time-lapse microscopy of endothelial cells after addition of medium or forskolin. Arrowheads indicate cell edges. See also the supplemental Videos. Graph: Heywood circularity factor calculated at 10-minute intervals for 60 minutes before and after forskolin treatment. (H) Endothelial cells treated with or without forskolin were attached to vitronectin-coated coverslips for 60 minutes and stained with rhodamine-phalloidin (actin), anti-vinculin (vinculin), or anti-integrin αvβ3 integrin β3. Arrowheads indicate focal adhesions. Graph: number of vinculin-containing focal adhesions in cells treated with or without forskolin.