Cell survival and activation of Akt in epithelial cells adhering to laminin-511.

<p>(A) m-IC_Cl2 epithelial cells and (B) embryonic smooth muscle cells (SMC) were plated on uncoated dishes +/− EGF and insulin, and on dishes containing laminin-511 (LM-511) or laminin-111 (LM-111). The PI3K inhibitor wortmannin was added where indicated. Cell lysates were analyzed by western blotting for phosphorylated Akt (P-Akt), Akt (pan-Akt) and actin. Note that activation of Akt is detectable in epithelial cells cultured on laminin-511 but not on laminin-111 (see quantification of the representative gel). No activation occurred in smooth muscle cells in the presence of laminin-511. In parallel, epithelial (A) and smooth muscle cells (B) were photographed by phase contrast microscopy on laminin-511 matrix (LM-511) and on laminin-111 (LM-111) with or without wortmannin (Wm). Cell spreading on laminin-511 (left pictures) versus laminin-111 (right pictures) was confirmed by flattening of the cells and reorganization of the cytoskeleton as probed with TRITC-phalloidin to visualize F-actin. (C) Representative immunoblots showing the expression of Akt (pan-Akt), Akt2 and Phospho-Akt (P-Akt) in E15.5 control (WT) and knockout (KO) intestines and quantification of two independent experiments as ratio between KO (grey bars) and WT (black bars) intestines. Data were normalized using actin.