Fig. S8

PGE2 affects hematopoietic progenitor function by enhanced cAMP production and subsequent phosphorylation events
(A – B) CFU-S₁₂ colony formation after transplantation of 500 purified KSL cells into irradiated recipient C57Bl/6 mice. Spleens were dissected at day 12 after transplantation and colonies counted.
(A) Effect of in vivo treatment of lethally irradiated recipient mice with indomethacin (2.5 mg/kg, i.p., q.o.d.), BIO (50μg/kg), or both, on CFU-S₁₂ colony formation; BIO treatment significantly increased colony formation, which could be inhibited by concurrent treatment with indomethacin (* statistically different from control and other treatments, t-test, p=0.015, n≥7).
(B) Effect of ex vivo treatment of purified KSL cells with Dkk1 (10ng/ml), dmPGE2 (10μM), forskolin (Fors; 1μM), cholera toxin (Chol Tox; 0.5μM) and/or a combination of drugs as indicated on CFUS₁₂ activity. Inhibition of wnt activity by Dkk1 reduced colony formation. This effect could be rescued by both dmPGE2 and direct cAMP stimulation by forskolin or cholera toxin; * statistically significant vs. control, ANOVA, p<0.05, n=7- 10.
(C) cAMP luminescence assay in isolated murine bone marrow cells; luminescence intensity is inversely related to cAMP levels. Exposure of bone marrow with dmPGE2 and forskolin for 15 minutes had dose-dependent enhancing effects on cAMP levels (mean ± SD, n=3).
(D) Western blot analysis of homogenized murine bone marrow cells following ex vivo drug treatment. Total β-catenin levels increased in a time-dependent fashion following exposure to dmPGE2 (10μM), and decreased in response to indomethacin (10μM). The response of phospho-β-catenin(S675) and phospho-GSKβ(S9) to drug treatment was also time dependent, and preceded effects on total β-catenin.