Figure 4

Effects of Shb deficiency on hematopoietic colony formation ability and cytokine expression in BCR-ABL-transformed bone marrow. (a) Bone marrow cells were plated on M3434 semisolid medium containing cytokines supportive of myeloid and erythroid colony growth. The number and types of colonies were determined on day 10 of culture [Granulocyte Erythroid Monocyte Megakaryocyte (GEMM), Burst-forming Unit Erythroid (BFU-E), Granulocyte Monocyte (GM), Monocyte (M), Granulocyte (G)]. Data are means based on 3 mice of each genotype. (b) A gradient of 0, 0.1 and 1 ng/ml of GM-CSF was used to evaluate cytokine responsiveness in leukemic bone marrow cells. Results are presented as percentage of the highest GM-CSF dose. Means ± SEM are representative of 3 mice of each genotype. (c) The expression levels of various hematopoietic cytokines were determined by semi-quantitative real-time RT-PCR in samples isolated from c-Kit⁺ leukemic bone marrow. All C_t values were normalized to β-actin and Shb knockout samples were related to corresponding wild type values. Means are presented as 2^-ΔC_t ± SEM to demonstrate fold change in mRNA content. Data are based on 6 mice of each genotype from 2 independent experiments for c-Kit⁺ cells and 3 mice of each genotype from 1 experiment for unfractioned bone marrow. *denotes p < 0.05 respectively as determined by Student’s t-test.