Fig. 3

PD0325901 prevented formation of the primitive streak and inhibits EpiSC self-renewal. a Western blot analysis showed PD0325901 (PD, 1 μM) prevented expression of PS marker T in the presence of CHIR99021 (CHIR, 3 μM) in EpiSCs cultured in N2B27 for 24 hours. b PD0325901 prevented expression of PS marker T and endoderm marker FOXA2 in the presence of Activin A (10 ng/ml) in EpiSCs cultured in N2B27 for 24 hours. c Real-time PCR showed that PD0325901 inhibited differentiation of PS even in the presence of Activin A or CHIR99021. d Immunostaining showed that PD0325901 inhibited both OCT4 and NANOG expression, whereas SB431542 (2 μM) only inhibited NANOG expression in EpiSCs differentiated in N2B27 for 24 hours. Bar, 100 μm. e PD0325901 inhibited both OCT4 and NANOG expression detected by western blot analysis. f Oct4 knockdown promoted PS differentiation in EpiSCs cultured in N2B27 medium for 24 hours. Pluripotent markers, Oct4 and Nanog; primitive streak markers, T and Mixl1; endoderm marker, Foxa2; neuroectoderm markers, Sox1, Sox2 and Pax6. AA Activin A, NC negative control, DMSO dimethyl sulfoxide, EpiSC epiblast stem cell, FGF fibroblast growth factor, T brachyury, ERK extracellular signal-regulated protein kinase, OCT4 octamer-binding transcription factor 4, FOXA2 forkhead box protein A2, SOX2 sex determining region Y-box 2, Mixl1 mix paired-like homeobox, Pax6 paired box 6