Fig. 5.

The crucial time period for H3K27me3 is within the first 4 days of differentiation. (A) Diagram showing the drug treatment plan for differentiation and re-plating of cells. Light gray circles indicate the cells used for the beating heart assay (F), other colors correspond to the bar charts quantifying AP staining (C,D). (B) Images of AP staining from DMSO- or GSK343-treated cells re-plated in ES conditions for 5 days after 8 or 14 days of EB differentiation. A greater proportion of cells that start in GSK343 treatment stain pink, indicating that the cells have reverted to an ESC phenotype. (C,D) Quantification of AP staining from cells treated with DMSO or GSK343 re-plated after 8 days (C) or 14 days (D) of EB differentiation performed in triplicate. Time of the treatment switch is indicated in the key and corresponds with colors in A. Data are mean±s.d. *P<0.05, **P<0.005 (unpaired two-tailed Student's t-test). (E) Unsupervised clustering of RNA-seq average expression of cells re-plated for 5 days after 14 days of EB formation. Cells that were treated with GSK343 have gene expression profiles that cluster with average expression from WT ESCs. All 7190 PRC2 target genes are shown. (F) Quantification of beating heart assay with GSK343- and DMSO-treated cells. Cells were differentiated as EBs for 4 days and then plated into differentiation media. Blue indicates the number of EBs that formed beating cells and orange indicates the number that did not. P-values were calculated using a two proportion z-test.