Fig. 1. High SOX2 levels inhibit WNT-induced differentiation of epiblast-like cells.
a, Schematic of mESC differentiation. F, FGF; L, LGK974; C, CHIR99021; 2i, CHIR99021 + PD0325901. b, To generate SOX2-TetON cells, a Dox-inducible SOX2 transgene was incorporated into the HPRT locus of an mESC line constitutively expressing the rtTA gene from the Rosa26 locus before ablation of endogenous SOX2 expression by gene editing. c–e, SOX2-ON and SOX2-OFF cultured in FL medium downregulate Nanog (c), upregulate FGF5 (d) and maintain Pou5f1 expression (e). f, SOX2 levels are negatively correlated with BRA expression in SOX2-ON cultured in FLC medium. g, Measurement of relative mRNA expression by PCR with reverse transcription (RT–PCR) shows that the kinetics of T/Bra induction is unaltered in SOX2-OFF cultured in FLC medium compared with CEpiLC controls. Each datapoint represents an individual biological replicate. Bars denote mean ± standard error of the mean (s.e.m.). P values calculated for differences of mean expression by two-tailed Students t-test are shown. n = 4 for both CEpiLC and SOX2-OFF at 60 h. n = 30 for CEpiLC and n = 18 for SOX2-OFF at 72 h. h, Representative plot from flow cytometry analysis shows that the distribution of T/BRA levels is unaltered in SOX2-OFF cultured in FLC medium compared with CEpiLC controls (arbitrary units). Source numerical data are available in source data.