Fig. 4. Integrin-β1-null endoderm cells fail to adhere and/or spread on BM substrates but can spread on vitronectin; vitronectin upregulates integrin β3 and induces GATA4 nuclear translocation. (A) Live phase micrographs show normal (WT) and integrin-β1-null (β1–/–) endoderm cells cultured on fibronectin for 16 hours. The cells were fixed and immunostained for paxillin, laminin 1 chain (Lm 1) and Dab2. F-actin was stained with rhodamine-phalloidin. (B) Endoderm cells were plated on various ECM substrates for 1 hour and adherent cells were quantified by crystal-violet staining and spectrophotometry. n=4 for each group. (C) Endoderm cells collected from the conditioned medium were fixed and immunostained for the apoptosis marker cleaved caspase-3. Positive cells were counted and plotted as a percentage of total cells examined. [n=326 for the wild-type (WT) and n=308 for integrin-β1-null (β1–/–) cells]. (D) Integrin-β1-null endoderm cells were grown on vitronectin (Vn)-coated coverslips for 2 days and immunostained for integrin β3, integrin v, paxillin, laminin 1 and Dab2. (E) The expression of integrin β3 was analyzed by FACS in integrin-β1-null endoderm cells before and after 2 days of culturing on vitronectin. (F) Integrin-β1-null endoderm cells were cultured on vitronectin, and GATA4 levels in cytosolic and nuclear fractions were assayed by western blotting at designated time points. The intracellular distribution of GATA4 was also analyzed by immunostaining. Vitronectin induced GATA4 nuclear translocation.