Figure 2.

WNT Pathway Activation Is Required for Primitive Streak Commitment and Endoderm Differentiation

(A) Immunofluorescence staining of anti-OCT4 (green) and anti-ABC (red) on D0 and D1 differentiation cultures performed as described in Figure 1, counterstained with DAPI (blue) for nuclei. Distribution of fluorescence intensity across a single representative cells was measured and plotted, demonstrating nuclear localization of ABC at D1. Scale bar represents for 100 μm.

(B) ChIP-qPCR analysis comparing D1 differentiation cultures with and without the Wnt agonist CHIR99021 (Chir) on primitive streak genes (T and EOMES) and endodermal genes (FOXA2 and SOX17). (i) and (ii) anti-OCT4, (iii) and (iv) anti-EZH2, (v) and (vi) anti-SUZ12, (vii) and (viii) anti-H3K27me3. Sites used for ChIP-qPCR are indicated in kilobases (K) from the transcription start site. Data shown are an average from three independent experiments. Statistical significance represented as ^∗p < 0.05.

(C) Flow cytometry analysis on D0 and D1 differentiation cultures with or without Chir addition, examining SOX2, OCT4, EOMES, and T expression.

(D) QRT-PCR analysis of D1 differentiation cultures with or without Chir addition.

(E) Immunoprecipitation of nuclear extracts from hES cells and D1 differentiation cultures with anti-OCT4 antibody and blotted with (left) anti-β-CATENIN and anti-OCT4 antibodies or (right) anti-active β-Catenin and anti-OCT4 antibodies; 1% of total lysate was run in the input lanes. Immunoprecipitation, IP; skipped lane, −; input, INP.