Fig. 6. Stabilized ß-catenin is insufficient for induction of primitive streak-associated gene expression. (A) A2lox.sßcat ES cells transiently transfected with the SUPER8xTOPFlash (TOP) or SUPER8xFOPFlash (FOP) reporters were left untreated (no treatment), or treated with either 1 µg/ml doxycycline (DOX) or LiCl for 18 hours. Whole cell lysates were prepared and luciferase activity measured. Shown is the luciferase activity normalized to a co-transfected Renilla luciferase construct driven by the CMV promoter as previously described (Ranganath et al., 1998). (B) A2lox.sßcat ES cells were differentiated in SRM alone or in the presence of Dkk1 (+Dkk1) and of the indicated concentration of doxycycline (DOX). Dkk1 was added at the initiation of differentiation. Doxycycline was added at day 1 of differentiation. Cells were harvested at day 3.5 of differentiation and evaluated for expression of the indicated genes by RT-PCR. Expression found in ES cells differentiated in SCM without doxycycline represent positive controls.