RA could not bypass the requirement for ERK signaling in neural specification. Monolayer differentiation of 46C ESCs in RA-treated cultures (N2B27 + 10-8 M RA) exposed to FGF receptor tyrosine kinase inhibitor SU5402 (5 μM), MEK1/2 inhibitors PD184352 (4 μM) or PD0325901 (4 μM), or to equivalent amounts of DMSO diluents. A, Cultures of monolayer differentiation, shown in phase contrast or stained for Sox1GFP; B, Typical FACS profile of Sox1GFP expression; C, Percentage of Sox1GFP+ cells scored by FACS; D, Cultures of monolayer differentiation, shown in stained for Oct4 or combination of Oct4 and Hoechst; E, Western blot analysis of Oct4 at day 5 under monolayer cultures; F, Western blot analysis of phosphorylated ERK1/2 and ERK1/2 at 12 h under monolayer cultures; G, RT-PCR for Elk1, one of the targets of ERK signaling; H, Q-PCR for Elk1 during monolayer differentiation. *, p < 0.05; **, p < 0.01. Scale bars: A, 40 μm; D, 80 μm.