Neural Differentiation Is Inhibited through HIF1α/β-Catenin Signaling in Embryoid Bodies

Figure 4

HIF1α attenuates neural phenotype by the stabilization of β-catenin in EBs. (a, A) The protein level of PAX6, BLBP, and TUJ1 in control (CTR) and β-catenin-transfected (βCAT) 5-day-old EBs (5d) derived from wild-type and Hif1α-deficient ESC. (a, B) Densitometric analysis and quantification (one-way ANOVA; for PAX6, for BLBP, and for TUJ1). (b, A) Western blot analysis of control (CTR) and Hif1α-transfected (HIF1α) 5-day-old EBs showing level of phosphorylated β-catenin. (b, B) Densitometric analysis is based on at least 5 independent experiments (one-way ANOVA). (c, A) Immunoprecipitation analysis from nuclear fraction of 2-day-old EBs. The analysis was performed using either IgG (isotype control) or HIF1α antibody as an input (“bait”), and β-catenin was detected afterwards (“prey”) as well as HIF1α in the magnetic bead fraction. (c, B) LaminB and β-actin were used as loading controls for nuclear and cytoplasmic fractions, respectively. Cytoplasmic β-catenin level does not differ between the two lines. CTR versus βCAT samples (a, A) as well as HIF(IN) versus IgG(IN) samples (c, A) were loaded on the same gel, but not directly beside each other. All procedural conditions and settings, including exposure time, were the same for all samples.

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