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Fig. 3. Interactions between FGF and Activin signalling pathways in hESCs. (A) Real-time quantitative PCR (QPCR) analysis of Oct-4 and Cripto expression in hESCs grown for four days in CDM supplemented with Activin (10 ng/ml) or FGF (40 ng/ml). Tra-1-60 positive and negative cells were sorted using FACS to separate undifferentiated and differentiated cells. Normalised Oct-4/PBGD (left panel) and Cripto/PBGD (right panel) mRNA levels were measured by QPCR. hESCs grown on feeders and differentiated cells from EBs were used respectively as positive and negative controls. Similar results were obtained in three independent experiments. (B) Effect of Cripto on hESCs grown in feeder-free conditions in CDM. Wild-type hESCs were grown for 7 days in CDM in the presence or absence of Cripto, Activin and FGF and in the presence or absence of the SB and SU inhibitors. FACS was used to determine the fraction of Tra-1-60 expressing cells. Values represent the mean and standard deviation of three separate experiments. (C). Western blot analysis of Smad2/3 phosphorylation in hESCs grown in feeder-free conditions. hESCs were grown for 6 days in CDM supplemented with 10 ng/ml Activin and 12 ng/ml FGF2 without feeders. Then hESCs were grown in different culture conditions for 2 hours. HepG2 cells were used as a positive control. Nuclear proteins were extracted and the expression of the phosphorylated form of the Smad2 protein was analysed using western blot (upper panel). Alternatively, total cellular extracts were used to confirm that expression of the Smad2 protein was the same in all the conditions (lower panel). Similar results were obtained with hESCs grown on feeders (data not shown). (D) Western blot analysis of Smad1/5/8 phosphorylation in hESCs grown in feeder-free conditions in the presence or absence of BMP4 (100 ng/ml). hESCs were grown for 6 days in CDM supplemented with 10 ng/ml Activin and 12 ng/ml FGF2 without feeders. Then hESCs were grown in different culture conditions for 2 hours. Nuclear proteins were extracted and the expression of the phosphorylated form of the Smad1/5/8 protein was analysed using western blot (upper panel). Alternatively total cellular extracts were used to confirm that expression of the Smad2 protein was the same in all the conditions (lower panel). (E) Recombinant BMP4 activates the Tlx2-lux reporter in feeder-free conditions. H9 cells were transiently transfected with the Tlx2-lux vector. After transfection, cells were incubated 48 hours in the absence (Neg) or in the presence of the SU inhibitor (10 µM) or BMP4 (100 ng/ml) alone or combined with FGF2 (40 ng/ml) or SU inhibitor (10 µM). Normalised luciferase activity is expressed as the mean ± SD from three informative experiments.
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