PDGF-BB–induced SMC migration implicates Shh-mediated PI3Kγ activation. (A) SMCs were pretreated for 30 minutes with Wortmannin, a pan PI3K inhibitor (Wort; 100 nmol/L), or AS-252424, a PI3Kγ selective inhibitor (AS; 100 nmol/L), then stimulated for 15 minutes with PDGF-BB (10 ng/mL) or Shh (1 µg/mL). Proteins were extracted and processed by western blot before Akt and phosphorylated Akt (p-Akt) detection. Kinetics of Akt phosphorylation in the presence or absence of the Hh blocking antibody 5E1 (Anti-Hh ab; 1.5 µg/mL) and control IgG1 (B) and in the presence or absence of the PI3Kγ selective inhibitor (AS) and dimethylsulfoxide (DMSO) (C) using western blot (left panels). Secondary Alexa 700-nm coupled antibodies (Invitrogen) and Alexa 800-nm coupled antibodies (LI-COR) were resolved with an infrared system (OdysseyR, LI-COR). Quantifications of band intensity performed with Odyssey software are shown (right panels). Immunoblots shown are representative of 3 independent experiments. (D) Basal and PDGF-BB–induced SMC migration in the presence or absence of PI3Kγ inhibitor (AS; 100 nmol/L) or Wortmannin (Wort; 100 nmol/L) or after transduction with empty (ad empty) or PI3Kγ inactive form (ad KR) adenoviruses. (E) SMC motility was measured after 72 hours in wound-healing assay and after 15 hours in random motility assay with or without stimulation by recombinant Shh (1 µg/mL) and in the presence or absence of PI3Kγ inhibitor (AS; 100 nmol/L). Data represent means ± SD from 3 independent experiments performed in triplicate. *P < .05; ***P < .0005.