Supplementary Figure 6: Signalling pathways in the decision between self-renewal and differentiation.

(A) The timing of insulin addition determines its impact on differentiation. NanogGFP ESCs were differentiated to PrE for 6 days. Insulin at 12.5 ng μl⁻¹ was added for the time shown. Cells were assessed for PrE differentiation by staining for PDGFRA and analysing by flow cytometry. Flow cytometry dot plots show quadrants based on NanogGFP (x-axis) and PDGFRA (y-axis). Dot plots are representative of 3 differentiations. (B) Response of cell growth to insulin in PrE differentiation. Total cells from three different E14TG2A derived ESC cell clonal lines were counted at the end point of differentiation. The graph shows that insulin has a large impact on cell number. N = 3 biological replicates, data is the mean and errors bars are plus and minus s.d. Statistical significance was calculated by t-test and the P value was 0.013. (C) Response of ESCs to insulin signalling. Two pairwise comparisons were carried out based on the experiment described in Fig. 5g. The first is shown in Fig. 5i. The second compared PDK1 mutants in NACL versus 0-h (insulin withdrawal). For both comparisons, the list of genes significantly stimulated by insulin (downregulated upon insulin withdrawal) was compared via a Venn diagram (generated via http://bioinfogp.cnb.csic.es/tools/venny). (D) Quantification of western blot analysis for Gsk3 phosphorylation (S9/S21) in cells with or without insulin. N = 3 independent clones. Error bars represent the s.d. of, P = 0.022 (t-test). (E) ESCs were cultured in 2i/LIF and NACL for 5 passages, then assessed for gene expression by qRT–PCR. Bars show the mean (n = 3 biological replicates), error bars ± s.d., P = 0.0030 (t-test). (F) NANOG protein levels in different ESC culture conditions. Quantification of Western blot analysis for NANOG expression in E14TG2A ESCs at p2 and p4 in the indicated culture conditions. Error bars represent the s.d. for n = 3 experiments. (G) Karyotype of NACL cultured ESCs. The chromosome count for two high-passage (>10) NACL cultures is shown. The average for both is 40, a normal count in mouse ESCs. (H) Circles show Alkaline Phosphatase (AP) staining of ESCs plated in the same density into 2i/LIF or NACL. nEnd cultures are shown as AP negative controls. Left panel: Whole stained 6 well plate. Right panel: images of individual colonies in NACL and 2i/LIF. Both colonies have high levels of AP staining, and tight circular morphologies, scale bars 800 μm. Images are representative of 6 experiments, and images of individual colonies are representative of 6 different photographed fields. (I) Transcriptomes of three self-renewing cell types. RNA expression as determined by microarray for RNA. The average of three replicates is shown, fold change cut off = 2, FDR threshold ≤ 0.05. (J) PrE (H +) and Epi (H −) primed ESCs have different growth efficiencies in different ESC medias. HexVenus ESCs were sorted based on HexVenus expression (top and bottom 25%) and plated into 2i/LIF or NACL for 7 days. These cells were then counted. Bars show the mean (n = 3 independent sorting experiments), error bars show s.d. P values calculated by t test, from top to bottom: 0.031, 0.027, 0.008, 0.024, 0.046.