Fig. 5. Involvement of phosphatidylinositol-3 kinase in the TGF-ß-induced activation of actin filament system. (A) PC-3U cells were serum-starved in 1% FBS for 12 hours, treated or not for 1 hour with 10 µM of the phosphatidylinositol-3 kinase inhibitor LY204009, before stimulation with 10 ng/ml of TGF-ß for 30 minutes or 24 hours. Filamentous actin was visualized by TRITC-labeled phalloidin. Arrowheads indicate cells with lamellipodia (30 minutes) and stress fibers (24 hours). Bar, 20 µm. (B) PC-3U cells were starved in 1% FBS for 12 hours and stimulated with 10 ng/ml of TGF-ß for different time periods as depicted in the panel. The total cell lysates were subjected to SDS-PAGE followed by immunoblotting using specific Akt antibodies. In the upper and lower panels, the phosphorylated and the nonphosphorylated forms of Akt are shown, respectively. (C) PC-3U cells were starved in 1% FBS for 12 hours, pretreated for 1 hour with or without 10 µM of the phosphatidylinositol-3 kinase inhibitor and stimulated with 10 ng/ml of TGF-ß for 30 minutes and 24 hours. The total cell lysates were subjected to SDS-PAGE followed by immunoblotting with specific Akt antibodies. (D) PC-3U cells were starved in 1% FBS for 12 hours, pretreated for 1 hour with or without 10 µM of the phosphatidylinositol-3 kinase inhibitor and stimulated with 10 ng/ml TGF-ß for up to 48 hours. The total cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies specific for Smad2 phosphorylated on serine residues 465 and 467 or Smad2.