Increase in neural differentiation of precursor cells by GSK3β inhibitor (BIO) during days 4 to 6 in N2B27 medium. (a)–(f) 46C ES cells were cultured in N2B27 medium for 6 days. GFP expression was elevated by BIO treatment (0.75 μM) from days 4 to 6, whereas GFP expression was reduced by BIO treatment (0.75 μM) from days 0 to 3. (g) FACS analysis of Sox1-GFP expression during monolayer differentiation in N2B27 medium. (h) Proportions of Sox1-GFP expressing cells as determined by FACS. (i) RT-PCR analysis showed that Nanog and Sox1 expression were elevated by BIO treatment (0.75 μM) under nondifferentiation and neural differentiation conditions, respectively. Scale bars, 100 μm.