FIG. 6.

Oct4 is essential for the induction of Brachyury expression by Wnt/β-catenin signaling. (A) The expression levels of Brachyury in the same Oct4-knockdown P19 EC cells that are used for the experiments shown in Figure 2A. (B) The induction of Brachyury and Sp5 by BIO (2 μM) in the presence or absence of Oct4 in P19 EC cells. P19 EC cells are transfected with control or Oct4 shRNA plasmid for 24 h, and then cultured in the presence of puromycin with or without BIO for another 24 h, followed by qRT-PCR analysis. (C) Gene expression changes in ES cells in response to Oct4 knockdown by shRNA plasmid transfection. The data show gene expression levels in Oct4 shRNA plasmid-transfected relative to control shRNA plasmid-transfected ES cells. After 1 day of transfection, ES cells are cultured for another day in the presence of puromycin to enrich for transfected cells. (D) The induction of Brachyury and Sp5 by BIO in the presence or absence of Oct4 in ES cells. The experiment is conducted in the same manner as the one presented in (B). (E) Reduction of Oct4 protein in ZHBTc4 ES cells in response to tetracycline (Tet; 10 ng/mL) treatment for 24 h. Oct4 protein is detected immunocytochemically and the nucleus is observed with DAPI. (F) The induction of Brachyury by BIO in ZHBTc4 ES cells. ZHBTc4 ES cells are cultured with or without BIO and/or Tet for 24 h, followed by qRT-PCR. (G) The induction of Brachyury and Sp5 by BIO in the presence or absence of Nodal signaling inhibitor SB431542. P19 EC cells are cultured with or without BIO and/or SB431542 (2 μM) for 24 h, followed by qRT-PCR. (H) The induction of Brachyury by BIO in the absence of Tdgf1. P19 cells are transfected with Tdgf1-specific shRNA plasmid, cultured with puromycin for 24 h, and then treated with BIO for 24 h, followed by qRT-PCR. ES, embryonic stem. Color images available online at www.liebertonline.com/scd.