Epac-1/Rap-1 and its downstream signaling Akt and PLCε, but not Rac-1, mediate cilostazol-induced PGI<sub>2</sub> production.

<p>(A) Top, Representative immunoblots showing total Rap-1 expression and Rap-1 activation in HAECs. Middle, Effect of Rap-1-targeting siRNAs (iRap-1) or non-targeting siRNAs (iCt) on protein levels in HAECs. Bottom, Effect of Rap-1 knockdown on PGI<sub>2</sub> production in HAECs. HAECs were transfected with iRap-1, or with the iCT. Post-transfection HAECs were treated with vehicle or 30 μM cilostazol (n = 4; † <i>p</i> < 0.05 vs. vehicle <i>t</i>-test). (B) Top, Representative immunoblot showing PLCε expression in HAECs. Middle, Effect of PLCε-targeting siRNAs (iPLCε) or non-targeting siRNAs (iCt) on protein levels in HAECs. Bottom, Effect of PLCε knockdown on PGI<sub>2</sub> production in HAECs. HAECs were transfected with iPLCε, or with the iCT. Post-transfection HAECs were treated with vehicle or 30 μM cilostazol (n = 4;† <i>p</i> < 0.05, †† <i>p</i> < 0.01 vs. iCT, <i>t</i>-test). (C) Top, Representative immunoblot showing P-PDK1, PDK1, Akt, and P-Akt expression in HAECs. Bottom, Effect of the PI3Kγ antagonist, AS60520, on PGI<sub>2</sub> production in HAECs (n = 4, †† <i>p</i> < 0.01 vs. vehicle, <i>t</i>-test; ** <i>p</i> < 0.01 vs. 30 μM cilostazol alone, lower-tailed Williams’ test).</p>