Supplementary Figure 7: Analysis of signalling in self-renewing 2i/LIF, NACL and nEnd conditions.

(A) Graphs displaying the total expression level of different transcription factors, signalling molecules/kinases, their activated forms, and the ratio between these in the three medium conditions 2i/LIF (2i/LIF), NACL and nEnd, and the expression levels of GP130, GATA6, KLF4 and NANOG. All were normalized to the expression of Histone 3 in the corresponding sample, and then averaged (n = 3 biological replicates). Error bars indicate the s.d. in each medium condition shown in B. (B) Western blots characterizing expression levels of markers for self-renewing ESCs (NANOG, KLF4, gp130), differentiated PrE (GATA6, PDGFRα) and activated kinases downstream of LIF, FGF, WNT, NODAL/ACTIVIN and insulin signalling. Protein lysates were collected from three cell lines grown in the self-renewing conditions 2i + LIF and NACL (N2B27 + ActA + CHI + LIF), and from the same cell lines after >5 passages as differentiated primitive endoderm (nEnd). The number of passages in these 3 media conditions is given in the top (for example, HVMC p5 PrE: passage 5 of cell line HVMC in PrE conditions). Full blots with indicated sizes are shown in Supplementary Fig. 9.