Fig. 3. Effects of activin A on GFP-Bry expression and endoderm induction in EBs. (A) Kinetics of GFP-Bry expression in EBs differentiated in SF cultures in the presence (+activin, 100 ng/ml; open circles) or absence (–activin; closed squares) of activin. (B) Temporal analysis of gene expression in EBs differentiated in the presence (100 ng/ml) or absence of activin in SF cultures. C, controls; top six lanes, day 3 serum-stimulated EBs; lower three lanes, day 6 serum-stimulated EBs. (C) FACS analysis demonstrating the effect of different activin concentrations on GFP-Bry expression in day 6 EBs. (D) RT-PCR analysis demonstrating the effect of different activin concentrations on gene expression profiles in day 7 EBs. (E) Immunostaining demonstrating the presence of Foxa2 protein in day 6 EBs differentiated in the absence (0 ng/ml) or presence (3 ng/ml or 100 ng/ml) of activin. Pink color indicates Foxa2-positive cells.