Fig. 1. The undifferentiated state of hESCs is characterized by activation of SMAD2/3-mediated signal transduction and inhibition of SMAD1/5-mediated signal transduction. (A) Western blot analysis of H1 hESCs cultured under various conditions. Cells were cultured for 4 days in the presence of: nCM supplemented with 2 µM BIO, non-conditioned medium (nCM), MEF-conditioned medium (CM), CM supplemented with 25 ng/ml BMP4, and nCM supplemented with 25 ng/ml activin A. Membranes were probed with antibodies specific for phosphorylated (P) SMAD2, SMAD2/3, phosphorylated SMAD1/5, SMAD1/5, OCT3/4 and -tubulin (as a control for protein loading). (B) Immunofluorescence microscopy of BGN2 hESCs in the undifferentiated and differentiated state. BGN2 cells were cultured for 5 days in CM, in nCM, in nCM supplemented with 25 ng/ml activin A, and in nCM supplemented with 2 µM BIO. Cells were decorated with an antibody specific for SMAD2/3, as well as SytoxGreen nuclear counterstain. (C) Bright-field images of H1 hESCs cultured in conditions described in A. Scale bars: 50 µm.