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Fig. S1. Endogenous retinoid levels differentially affect the expression of pluripotency genes. (Upper) Experiment schematic. ES cells were differentiated for two days in standard N2B27 media (+Retinyl) or N2B27 lacking (−Retinyl) retinyl acetate, then analysed by quantitative RT-PCR (qRT-PCR). (Lower) In −Retinyl conditions, levels of transcripts for Rex1 and Nanog are reduced, but Oct4 levels remain at similar levels compared to +Retinyl. Results are averages ±s.e.m. from three experiments performed in triplicate.
Fig. S2. PI3 kinase activity is not required for dP-Erk induction by RA. Cells were plated under differentiation conditions. Twelve hours later, cells were washed and re-fed with N2B27, incubated for 1 hour with vehicle or the PI 3-kinase inhibitor PI103 (Hayakawa et al., 2007; Raynaud et al., 2007) 0.1 mM, from Division of Signal Transduction Therapy (DSTT), University of Dundee, UK, then treated with 50 nM RA for a further 8 hours. Cells were then lysed and samples immunoblotted with the indicated antibodies. PI103 treatment cannot prevent the activation of Erk caused by RA.
Hayakawa, M., Kaizawa, H., Kawaguchi, K., Ishikawa, N., Koizumi, T., Ohishi, T., Yamano, M., Okada, M., Ohta, M., Tsukamoto, S. et al. (2007). Synthesis and biological evaluation of imidazo1,2-apyridine derivatives as novel PI3 kinase p110alpha inhibitors. Bioorg. Med. Chem.15, 403-412.
Raynaud, F. I., Eccles, S., Clarke, P. A., Hayes, A., Nutley, B., Alix, S., Henley, A., Di-Stefano, F., Ahmad, Z., Guillard, S. et al. (2007). Pharmacologic characterization of a potent inhibitor of class I phosphatidylinositide 3-kinases. Cancer Res. 67, 5840-5850.
Fig. S3. The effect of RA treatment on dP-Erk levels is inverted after day 1 of differentiation. (Upper) Experiment schematic. Vehicle (DMSO) or RA was added to differentiating ES cells at each day of differentiation for 24 hours. Cells were analysed by flow cytometry or immunoblotting. (Lower) Addition of RA for the first 24 hours results in increased levels of Erk1/2 phosphorylation, whereas at all later time points tested RA results in a reduced Erk1/2 phosphorylation. Addition of RA increased the proportion of Sox1-GFP expressing neural progenitors and induced expression of the RA-responsive gene Crabp1.
Fig. S4. Addition of RA does not reduce Fgf receptor expression or induce intracellular inhibitors of the Fgf pathway. Cells were treated during day 2 of differentiation with vehicle (DMSO) or RA, then analysed by quantitative RT-PCR (qRT-PCR) for expression of the Mapk phosphatases Dusp1, Dusp2 and Dusp6 (A), the Fgf receptors Fgfr1, Fgfr2 and Fgfr3 (Fgfr4 was not detected; B), or Sprouty2 (C). RA treatment does not cause any significant change in the expression of these genes. Results are averages ±s.e.m. from two independent experiments performed in triplicate.
Fig. S5. Vegfr inhibition does not promote differentiation. Sox1-GFP/46C cells were treated during the second day of monolayer differentiation with vehicle (DMSO), RA, the small molecule inhibitor PD173074 or the selective Vegfr inhibitor KRN633 (Nakamura et al., 2004) (Calbiochem) at the concentrations shown. Following 24 hours of treatment, the cells were trypsinised and analysed for Sox1-GFP by flow cytometry. KRN633 does not mimic the effect of RA or PD173074, suggesting that PD173074 acts via its ability to inhibit Fgfr and not via Vegfr inhibition.
Fig. S6. RA treatment on day 2 does not alter cell cycle stage distribution. Cells were treated during the second day of differentiation with vehicle (DMSO) or RA, then fixed in ethanol, stained with propidium iodide and analysed by flow cytometry for DNA content. There is no difference in the proportion of cells in each cell cycle phase when RA is added for 24 hours at day 2.