Fig. S1.

Smad7 promotes self-renewal and inhibits differentiation of ESCs. (A) Smad7 is down-regulated during EB formation in CGR8 cells. Cell lysates were subjected to Western blot analysis to examine protein levels of Smad7, Oct4, Sox2, Nanog, and GAPDH (as a negative control) with appropriate antibodies. (B) Differentiation markers are up-regulated during EB formation in CGR8 cells. Total RNAs were subjected to qRT-PCR to examine mRNA levels of Cxcl12, Brachyury/T, and Foxa2. Data are shown as mean ± SEM; n = 3. (C) Dox induces an approximately two-fold increase in Smad7 mRNA in SFB–Smad7–tet-on CGR8 cells. Wild-type and SFB–Smad7–tet-on CGR8 cells were cultured in the absence or presence of 1 μg/mL Dox for 72 h. Total RNAs were extracted from cells and subjected to qRT-PCR to examine expression levels of Smad7. Data are shown as mean ± SEM; n = 3. *P < 0.05. (D) Dox induction of Smad7 gives a moderate increase in total Smad7 protein in SFB–Smad7–tet-on CGR8 cells. Wild-type and SFB–Smad7–tet-on CGR8 cells were cultured in the absence or presence of 1 μg/mL Dox for 72 h. Cells were subjected to Western blot analysis to examine the protein level of Smad7. (E) Dox treatment maintains the mRNA level of Smad7 during EB formation. SFB–Smad7–tet-on CGR8 underwent EB differentiation for 4 d in the absence or presence of 1 μg/mL Dox. Total RNAs were extracted from cells and subjected to qRT-PCR analysis to examine the mRNA level of Smad7. Data are shown as mean ± SEM; n = 3. *P < 0.05.