Fig. 3. Stimulation of endogenous ß-catenin signaling overcomes the inhibitory effects of high density on RA-treated cultures. Stimulation of endogenous signaling was mediated by treatment with Wnt3a-conditioned media and by overexpression of H2kd-E-cadherin. (A) HEK293 cells were transfected with TCF/LEF-luciferase and tested for luciferase activity after treatment with Wnt3a-conditioned media or after cotransfection of H2kd-E-cadherin with TCF/LEF-luc. H2kd-E-cadherin was able to increase luciferase activity, although Wnt3a treatment had a greater effect. (B) Changes in ß-catenin degradation were analyzed in ES cells after Wnt3a treatment. ES cells were induced by EB formation in high density and were either treated with control-conditioned media or with Wnt3a-conditioned media. Total cell lysates were extracted on day 2 of the differentiation (2 days Wnt3a treatment) and analyzed by western blotting (B). Treatment with Wnt3a-conditioned media reduced the amount of phospho-ß-catenin while not significantly altering the levels of total ß-catenin. Wnt3a treatment resulted in a decrease in the levels of phopshorylated ß-catenin signaling. ES cells were induced by EB formation at high density in either control-conditioned media (C) or Wnt3a-conditioned media (D), and differentiated by the 4-/4+ protocol. Wnt3a treatment resulted in many ß-tubulin 3-positive cells (D), whereas control media did not (C). Cells were stably transfected with an empty vector (E), H2kd-E-cadherin (F) or full-length ß-catenin (G), and induced by EB formation at high density using the 4-/4+ protocol. Overexpression of H2kd-E-cadherin and ß-catenin resulted in neuronal differentiation in high-density cultures, but transfection with empty vector did not. (C-G) All cells were fixed 3-4 days post-plating, and stained with anti-ß-tubulin 3 antibodies (green) and counterstained with Hoechst (blue).