Figure 2.

MK maturation of normal and MDS marrow in response to SU6656. (A) Primary human CD34 ⁺/CD38^lo cells were cultured in a 4-cytokine media for 10 days as previously described.⁷ After 10 days, cultures were washed and resuspended in serum-free media containing 35 ng/mL rhTPO. Cells were then cultured in the absence (vehicle) or the presence of 2.5 μM SU6656. After 72 hours, the samples were labeled with propidium iodide and analyzed by flow cytometry to detect ploidy classes. (B) Mononuclear cells from an individual with myelodysplastic syndrome were cultured in the presence of rhTPO (35 ng/mL) for 5 days in the absence or presence of SU6656 (2.5 μM). Cells were treated with 0.1% SDS, RNase, and propidium iodide. Nuclear ploidy was determined by flow cytometry and plotted on a semilog scale (1 × 10⁶ events/sample). (C) MDS primary cell cultures were cytospun onto glass slides and stained with Wright-Giemsa. The photographs show representative fields of preparations at × 50 magnification. (D) Cell lysates were generated from MDS primary cells under each culture condition. Lyn was immunoprecipitated with anti-Lyn antibody (Santa Cruz Biotech, Santa Cruz, CA), analyzed by Western blot, and probed with antiphospho-Src (Tyr416) (Cell Signaling). Blots were stripped and reprobed with appropriate antibodies to ensure an equal amount of protein in each lane. (E) Purified active Aurora kinase B (lane -: no Aurora kinase B) was preincubated with SU6656 (0-2 μM) prior to the addition of the substrate (dephosphorylated histone H3). Histone H3 phosphorylation detected by Western blotting using a phospho-specific H3 antibody. (F) Aurora B kinase was immunoprecipitated from UT-7/TPO lysates, and kinase activity was assayed using purified histone H3 as a substrate. Phosphorylation was detected by blotting with phospho-specific histone H3 antibody. Blots were reprobed with antibody to histone H3. Control lane contains purified histone H3 alone. IB indicates immunoblot.