Supplemental Material

This article contains the following supporting material:

• Figure 1 - RT-PCR analysis showed the expression pattern of FGF family members, IGF, EGF, and FGFR1 and FGFR2 during RA-induced P19 cell neural differentiation. The white dash lines indicated the RA induction stage.

• Figure 2 - Endodermal markers, AFP and GATA4, were expressed during monolayer P19 cell differentiation. P19 cells were treated with 10^-6M RA, 10 ng/ml FGF8, or RA plus FGF8 for two days, and cells were cultured in N2 medium for another 3 days, then were immunostained. Scale bar: 100 μm.

• Figure 3 - FGFR3-Fc could reduce the RA-induced P19 cell neural differentiation. (A-C) Immunostaining of Oct4^-Sox⁺ cells showed that 100 ng/ml FGFR3-Fc could significantly inhibit the expression of Oct4^-Sox⁺ cells in RA-induced P19 cell aggregates, while other FGFR-Fcs could not. Scale bar: 100 μm.

• Figure 4 - FGF8 inhibited Smad1 nuclear translocation through phosphorylating the linker region of Smad1. (A) P19 cells were cultured in serum containing and serum-free medium, and treated with FGF8, U0126, BMP2. Immunostaining was used to detect subcellular translocation of endogenous Smad1 protein. (B) P19 cells were cultured in serum-free medium, and treated with FGF8 (10 ng/ml) and U0126 (5 μM). Western blot was used to detect Erk1/2 and phospho-Erk1/2 expression. (C) FGF8 induced the phosphorylation of WT-Smad1 and CM-Smad1, but not LM-Smad1 in transfected HEK293T cells (upper panel). Smad1 expression was examined by anti-Flag immunoblotting (lower panel). (D) In P19 cells, ca-BMPR-IB dependent Vent2-luciferase reporter activity was severely inhibited by FGF8. The inhibition of reporter gene expression by FGF8 was rescued by U0126, suggesting FGF8 functions through Erk1/2. (E) FGF8 inhibited the ca-BMPR-IB induced Vent2-luciferase expression in WT-Smad1 transfected P19 cells, but not in LM-Smad1 transfected P19 cells. CM-Smad1 served as the negative control. Scale bar: 5 μm.