Extended Data Fig. 3: ESCs lacking MLL3/4 enzymatic activities can differentiate towards the three germ layers, but show defects in cardiomyogenesis.
a, b, Immunoblotting in f/f and KI cells at day 0 (D0), D4 and D10 of EB differentiation. Whole cell lysates (a) or histone extracts (b) were analyzed using indicated antibodies. Histone modification levels were quantified and normalized to H3. c, RT-qPCR analysis of primitive streak markers (T, Mixl1) at indicated time points. Data are presented as means ± s.d., n = 3 independent experiments. d,e, RNA-seq data of genes expressed in f/f or KI D4 EBs (d) and f/f or KI D10 EBs (e) were presented as scatter plots. Pearson correlation coefficient (R) and the corresponding two-sided P value are shown. The number of genes in each group is indicated in parentheses. f, Gene set enrichment analysis (GSEA) of 3,845 genes induced from ESC to D10 EB on developmental terms. Nom p-value was determined by a 1000-fold permutation test; FDR q-value was adjusted for multiple hypotheses testing. NES, normalized enrichment score. Statistically significant data are highlighted in bold. g, The percentage of beating EBs at indicated time points. Data are presented as means ± s.d.. The result is from 3 independent experiments with 24 EBs per group. h, Representative microscopic images of attached EBs at D12. Scale bar, 250 μm. i, Beating frequencies of EBs at D12 are presented as dot plots. 24 EBs were measured per group. Horizontal lines represent mean values. Statistical significance was determined by the two-tailed unpaired t-test. j, RT-qPCR analysis of the cardiac progenitor marker (Tbx5) and cardiac myofilament genes (Myh6, Myl2, Ttn) at indicated time points. Data are presented as means ± s.d., n = 3 independent experiments. k, Teratoma assay of f/f and KI ESCs. Representative histological sections of structures belonging to endoderm, mesoderm and ectoderm are shown. Scale bar, 100 μm.
