Bone marrow samples were fro zen in cryomolds and performed on 5 um sections. The slides were stained with Giemsa staining. Twenty five random high power fields from each useful handbook bone marrow sam ple were chosen and blindly quantified for histological examination. Murine Colony Forming Unit Assay The assay was performed as described previously. Colony forming unit granulocyte macrophage, burst forming unit/colony forming unit erythroid, and colony forming unit mixed were cultured in methylcellulose supple mented with fetal calf serum, 1% BSA, 0. 1 mM b mercaptoethanol, 3 IU/ml erythropoietin, 10 ng/ml granulocyte macrophage colony stimulating fac tor, 10 ng/ml interleukin 3, and 50 ng/ml SCF. Murine bone marrow cells were seeded in tripli cate and incubated for 7 days. Colonies were scored blindly.

Murine Colony Forming Unit Megakaryocytes Assay Murine bone marrow cells were cultured using the plasma clot culture method. The cul ture medium contains 1% deionized bovine serum albu min, 0. 34 mg CaCl2, 10% citrated bovine plasma, 100 ug penicillin, 50 ug streptomycin and IMDM with different concentrations of APS, TPO and IL 3 in a total volume of 1 ml. The cells were incubated at 37 C under 5% CO2 for 7 days and the number of CFU MK derived colonies was counted using acetyl choline esterase staining method after 7 days. The colonies were further stained with haematoxylin to count the CFU GM derived colo nies. A CFU MK colony was defined as a cluster of 3 or more AchE positive cells and a CFU GM colony was considered as a cluster of 40 or more cells.

Murine Bone Marrow Colony Forming Unit Fibroblast assay The assay was performed as described previously. Briefly, mouse bone marrow cells were seeded in 2 ml of IMDM with 10% FCS in tripli cates. Cultured cells were incubated at 37 C and 5% CO2 in a fully humidified atmosphere with or without APS for 9 days. Fibroblastoid colony forming cells assay were used to determine the number of bone marrow derived fibroblastoid. Briefly, adher ent cells were stained with Giemsa staining. The num ber of CFU F colonies was counted under a light microscope. An aggregate containing more than 10 fibroblasts were counted as a CFU F colony. Effects of APS and other cytokines such as fibroblast growth fac tor, platelet derived growth factor, or vascular endothelial growth factor were also examined using the CFU F assay.

Annexin V, Caspase 3, and Mitochondrial Membrane Potential Analyses of M 07e Cells by Flow Cytometry The assays were performed as described previously. Briefly, the megakaryoblastic cell line M 07e was maintained in IMDM Entinostat supplemented with GM CSF and 10% FCS. Apoptotic cell death was induced by cytokine and serum depletion. We added APS to the cultures and then the cells were incubated for 72 hours. Apoptotic cell death was examined using the annexin V FITC/PI, active Cas pase 3 PE, and JC 1 ApoAlert reagent kits according to the manufacturers instructions.