Fig. 6

ERK inhibition at epiblast-like stage promoted differentiation of neuroectodermal precursors from ESCs. a, b ESCs cultured in N2B27 medium for neuroectodermal precursor commitment on gelatin-coated dishes. a Activity of ERK1/2 from D0 to D5 detected by western blot analysis. b Relative intensity of western blot analysis from (a) analyzed by ImageJ software. c PD0325901 treatment on day 3 (48–72 hours) or day 4 (72–96 hours) increased Sox1 mRNA expression. **p < 0.01. d Immunostaining of neuroectoderm marker PAX6 and neural stem cell marker nestin. PD0325901 treatment (72–96 hours) increased expression of these markers. e, f FACS showed that PD0325901 treatment (72–96 hours) increased GFP-positive cells during 46C ESC neural differentiation. **p < 0.01. g GFP-positive cells from PD0325901 treatment (72–96 hours) were further cultured in N2B27 for 3 days and stained with TuJ-1 antibody for neuron detection. Bar, 100 μm. h Working model of PD0325901 on the commitment of epiblast into neuroectoderm precursor cells. PD0325901 inhibits the self-renewal by decreasing the expression of pluripotent genes, and blocks the formation of primitive streak by preventing the accumulation of β-catenin in the nucleus, thus promoting the differentiation of epiblast into neuroectoderm precursor cells. D day, DMSO dimethyl sulfoxide, ERK extracellular signal-regulated protein kinase, OCT4 octamer-binding transcription factor 4, Sox1 sex determining region Y-box 1, SOX2 sex determining region Y-box 2, GFP green fluorescent protein, Pax6 paired box 6