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Fig. 5. Interaction of Wnt and Notch signaling in modulating primitive
erythropoiesis from Flk1+ cells. Flk1 cells isolated from day
2.75 EBs generated from the indicated ES cell lines were cultured as
aggregates in serum free media containing hVEGF (10 ng/ml) and either Wnt3a
(100 ng/ml), DKK1 (300 ng/ml), Dox (2 µg/ml), 
-secretase inhibitor
(gSI; 2.5 µM) or DMSO (2.5 µl/ml), as specified. Hematopoietic potential
of the aggregates was assayed as described in the Materials and methods.
(A) Synergistic effects of Numb and Wnt3a on primitive erythroid
development from Flk1+ cells. (B) Synergistic effects of
-secretase inhibitor and Wnt3a on primitive erythroid development from
Flk1+ cells. (C) Effect of Wnt3a on Notch1-IC-induced
suppression of primitive erythroid development. (D) Effect of Numb
expression on DKK1-induced block in primitive erythropoiesis. DKK1 was added
to the reaggregation media after 6 hours of culture to allow time for Numb
protein to accumulate. (A-D) Error bars represent the standard deviation of
the mean of the number of colonies from n independent experiments (A,
n=6; B, n=4; C, n=5; D, n=3)
(**P-value<0.01;
***P-value<0.001). (E) The TOP/FOPflash reporter
assay demonstrates interaction between Wnt and Notch pathways. Error bars
indicate standard deviation of TOP/FOP values of triplicate electroporations.
Numbers above each bar represent actual TOP/FOP values.
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