Figure 7

EpCAM/ERas/AKT axis limits cardiomyocyte formation. (a) EpCAM-YFP and endogenous ERas interact in F9 teratoma cells and E14TG2α ESC transfectants expressing EpCAM-YFP or YFP. EpCAM-YFP and YFP were immunoprecipitated from stable F9 and E14TG2α ESC transfectants. Co-precipitated endogeneous ERas was detected with in immunoblots with specific antibodies. Immunoprecipitation (IP); immunoblot (IB) (n = 3 independent experiments). (b) Transcript levels of ERas were assessed from RNA-sequencing datasets generated from single cells at day E3.5–6.532 (left) and E6.5–7.7533 (right). Transcript levels are depicted as box-whisker plots with log10 normalised counts (adding a pseudocount of 1). See Fig. 1 and Methods for the approximate localization of cell types and for additional details on the datasets. (c) EpCAM and ERas mRNA expression was measured by quantitative PCR in pluripotent E14TG2 ESCs (d0) and EB (d2–14) (n = 3 independent experiments). (d) EpCAM and ERas protein expression was assessed by immunohistochemistry in E14TG2α ESC embryoid bodies (EB d2, d14) (n = 3 independent experiments). Scale bars represent 50 µm (d2) and 20 µm (d14). (e) AKT phosphorylation was assessed by immunobloting with the indicated antibodies in E14TG2α ESC expressing EpCAM-YFP or YFP, with or without treatment with insulin-like growth factor (IGF) (n = 3 independent experiments). (f) Quantification of AKT phosphorylation. Shown are mean n-fold changes normalized for AKT phosphorylation levels in control YFP transfectant cells (n = 3 independent experiments). (g) AKT phosphorylation was assessed by immunobloting with the indicated antibodies in wild-type E14TG2α ESC and knockout clone #114. Graph represents the quantification of AKT phosphorylation in wild-type E14TG2α ESC and knockout clone #114 (n = 3 independent experiments). (h) Shownare mean percent contracting EB of wild-type E14TG2α ESC (WT), pCAG, Flag-ERas and myrAKT transfectants (n = 6 independent experiments). (i) Shown are mean percent contracting EB of wild-type E14TG2α ESC (WT), single EpCAM (#58, #118) and double EpCAM/ERas (#58/11, #58/224, #188/11, #118/21) knockout clones (n = 3 independent experiments). Mean ± SEM; Student’s T-test (n = 2 groups) or One-Way ANOVA (n ≥ 3 groups); p < 0.05, **p < 0.01, ***p<0.001.