Fig. 4. Nuclear β-catenin signaling in TS cells in culture.

(A) Time-dependent accumulation and nuclear translocation of active β-catenin in response to recombinant Wnt3a protein (50 ng/ml) in differentiating TS cells. (B) Wnt3a induced c-Myc and PPARδ expression in differentiating TS cells. (C) DKK1 (1 μg/ml) and PKF115-584 (1 μM) blocked Wnt3a-induced cytoplasmic accumulation of active dephospho β-catenin in TS cells by 2 hour of cotreatments. Notably, even basal levels of nuclear β-catenin disappeared following PKF115-584 treatment. (D) Similar treatments with DKK1 (1 μg/ml) and PKF115-584 (1μM) downregulated Wnt3a-induced c-Myc and PPARδ expression in differentiating TS cells. (E) Dvl1-3 proteins in TS cells. While Dvl1 was only detected in the nucleus, Dvl2 and Dvl 3 were detected in both the cytoplasm and nucleus in response to Wnt3a. (F) Wnt receptors, Fzd 2, Fzd4, LRP5, LRP6, Kremen 1 and Kremen 2 in TS cells. Wnt3a facilitated nuclear import of Wnt receptor subtypes in differentiating TS cells, mimicking the finding in blastocysts during activation. Representative pictures of Western blotting analysis of Wnt family components in TS cells were presented in panels A-F. C, M or N, cytoplasmic, membrane or nuclear protein extraction.