FIGURE 5.

Activation of Epac with 007, partial silencing of Epac with shRNA, inhibition of cAMP phosphodiesterase with IBMX, or inhibition of RhoA by C3 toxin altered TGF-β1-induced EMT in EECs. The epicardial cells were treated with 250 pm TGF-β1 with/without 50 μm 007, 10 μm IBMX, or 3–5 μg/ml C3 toxin for 72 h followed by immunolabeling with antibodies for detection of SM22 and ZO1. TGF-β1 but not 007 treatment alone significantly increased the expression of SM22 protein (C versus B) and disrupted the cell-cell contacts as indicated with ZO-1 staining (G versus F), whereas these TGF-β1-induced changes were suppressed by treatment with either 007 (D and H), IBMX (L), or C3 toxin (P). Partial silencing of Epac1 by 45% ± 17 S.E., n = 8 (panel U), resulted in an increase in SM22 in both control and TGF-β1 treated EECs detected by immunohistochemistry (panels Q–T) consistent with Epac suppression of EMT (three independent experiments).