Fig. S7

Fig. S7 The PGE2/wnt interaction affects hematopoietic colony formation in murine ES cells
(A and B) ES cell hematopoietic colony formation assays were performed; cells were exposed to soluble modulators of the wnt pathway (10 ng/ml Wnt3A of 400 ng/ml Dkk1) and then incubated with indomethacin (20 μM), dmPGE2 (10μM), or forskolin (5 μM) as indicated. Colony forming units-Culture (CFU-E (erythroid), CFU-M (monocyte), CFUG (granulocyte), CFU-GM (granulocyte-monocyte), and CFU-GEMM (granulocyteerythroid- monocyte-megakaryocyte)) were scored on day 8-10 based on morphology. Averages ± SEM in the fold changes in total CFU-C number of each sample relative to control were calculated from at least six individual experiments.
(A) The positive effect of wnt activation on hematopoietic differentiation could be diminished by inhibition of prostaglandin synthesis with indomethacin. Wnt3A enhanced hematopoietic colony formation. Indomethacin (20μM) alone diminished colony number and inhibited the enhancing effects of Wnt3A (* statistically significant vs. control, t-test, p=0.002, ** statistically significant vs. indomethacin, p<0.001; n=6).
(B) Inhibition of wnt activity decreased hematopoietic differentiation and could be rescued by PGE2 or cAMP signaling. Inhibition of wnt activation by Dkk1 (10ng/ml) diminished colony number. dmPGE2 (10μM) and the cAMP activator forskolin (0.5μM) enhanced hematopoietic colonies. This negative effect of Dkk1 could be rescued by both dmPGE2 or forskolin, confirming the role of PKA in this process. (* statistically significant vs. control, t-test, p=0.005, *** statistically significant vs. Dkk1, p<0.046; n=4).
(C) Western blot analysis of ES cell lysates. Wnt3A and dmPGE2 treatment enhanced β- catenin levels, while indomethacin and Dkk1 had negative effects. Indomethacin diminished the enhancing effect of Wnt3A, whild dmPGE2 could enhance β-catenin levels after Dkk1 exposure.