Increased neurophenotype in Hif1α−/− EBs is associated with decreased nuclear β-catenin activity. (a) TOPflash assay performed in 2-day-old EBs (2d) to compare β-catenin nuclear activity in wild-type (+/+) and Hif1α-deficient (−/−) ESC cultivated in 20% (white bars) and 1% (black bars) of oxygen. Quantification is based on at least 3 independent experiments (one-way ANOVA). (b) Western blot analysis (representative picture) showing stabilization of HIF1α protein in 2-day-old EBs compared to ESC. Black arrow indicates band of HIF1α at expected molecular weight. (c) TOPflash assay in 2-day-old EBs (2d) (white bars for wt, black bars for Hif1α−/−) after transfection with either empty plasmid (CTR) or plasmid overexpressing mouse Hif1α (HIF1α). Quantification is based on at least 7 independent experiments (one-way ANOVA). (d) Relative gene expressions of Hif1α, Tcf1, Lef1, Tcf3, Tcf4, Axin2, and NeuroD1 detected in 2-day-old EBs derived from wild-type (white bars) and Hif1α-deficient cells (black bars) by real-time quantitative PCR analysis. Quantification is based on at least 3 independent experiments (t-test for each marker separately, Hif1α+/+ versus Hif1α−/−).