JCS02553 Supplementary Material

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• Supplemental Figure 1 - Fig. S1. Nuclear localisation of Smad2 protein in hESCs expressing the pluripotent marker SSEA-3. Co-Expression of SSEA-3 (green fluorescence, third and fourth panel) and Smad2 (red fluorescence, second and fourth panel) in hESCs grown in feeder-free conditions in CDM supplemented with Activin (10 ng/ml) and FGF2 (12 ng/ml). Nuclei are stained with Hoechst 33258 dye (blue fluorescence, first panel); Combined Smad2 and SSEA-3 staining are also shown as overlapping green (surface) and red (nuclear) signals (fourth panel). Bar, 50 mM.

• Supplemental Figure 2 - Fig. S2. Expression of pluripotency markers by hSF-6 cells grown for 5 passages (~ 25 days) in CDM in feeder free-conditions. hESCs were grown in CDM supplemented with Activin (10 ng/ml) and FGF2 (12 ng/ml) and expression of the pluripotency markers Oct-4, and Tra-1-60, SSEA-3, and SSEA-4 (green fluorescence) was analysed by immunofluoresence. Nuclei are stained with Hoechst 33258 dye (blue fluorescence). Bar, 100 mM.
• Supplemental Figure 3A
• Supplemental Figure 3B - Fig. S3. Consequences of Activin/Nodal/TGFb signalling inhibition on hESC pluripotency marker expression. (A) Inhibition of the Activin/Nodal/TGFb1 signalling pathway in hESCs on feeders by SB431542 (SB). hESCs were grown on feeders in the presence of SB (1 mM, 10 mM and 20 mM) for 5 and 10 days (1 passage). FACS was used to determine the fraction of Tra-1-60 expressing cells. Values represent the mean and standard deviation of three separate experiments. (B) Effect of the FGF receptor inhibitor SU5402 (SU) and the Activin/Nodal/TGFb receptor inhibitor SB on hESCs grown on Matrigel. Wild-type hESCs and Nodal-hESCs were grown for 10 days on Matrigel in feeder cell-conditioned medium supplemented with (+) or without (–) 4 ng/ml FGF and in the presence of SB (20 mM) or alternatively in the presence of SU (10 mM). FACS was used to determine the fraction of Tra-1-60 expressing cells. Values represent the means and standard deviations of three separate experiments.

• Supplemental Figure 4 - Fig. S4. Effect of SB431542 and SU5402 inhibitors on pluripotency marker expression of hSF-6 cells. hESCs were grown for 4 days on feeders in the absence (upper row) or in the presence of SB (middle row) or SU (bottom row) inhibitor. The level of differentiation was established by immunofluorescence to determine the expression of Oct-4 (green fluorescence). Nuclei are stained with Hoechst 33258 dye (blue fluorescence). Bar, 100 mM.