SCF-induced growth of Lyn-deficient progenitor cells is reduced compared with wild-type controls.

(A) Lyn-deficient mice do not express Lyn protein. Lysates were generated from wild-type (+/+) and Lyn-deficient (−/−) mast cells. Equivalent cell units were resolved by SDS-PAGE, transferred to Immobilon, and immunoblotted with antibody specific for murine Lyn. (B) Lyn-deficient progenitor cells (▪) proliferated more slowly than wild-type cells (♦) in response to SCF. Mononuclear cells from bone marrow from wild-type and Lyn-deficient mice were isolated using lymphocyte separation media. Lin⁻ progenitor cells were purified by negative selection, as described in “Materials and methods.” These cells were stained with antibody specific for murine c-Kit and were isolated using FACS. The resultant population of Lin⁻, c-Kit⁺(Lin^negKit^pos) progenitor cells was resuspended in QBSF-60 (serum-free) media supplemented with 100 ng/mL murine SCF, and the numbers of viable cells were counted each day. Data are representative of results from 3 experiments. (C) SCF-induced colony formation of Lyn-deficient progenitors (░) is reduced compared with normal cells (■). Wild-type and Lyn-deficient Lin^negKit^pos progenitor cells were resuspended in 1% methylcellulose with QBSF-60 media in the presence or absence of murine SCF (100 ng/mL). Five thousand cells were plated per 35-mm² Petri dish. After an 8-day incubation, the size and number of colonies were determined for each group. Data represent the mean and standard deviation of triplicate samples. No colonies formed in the absence of SCF. Small colonies were defined as those containing 10 to 25 cells, medium colonies as those containing 25 to 100 cells, and large colonies as those containing 100 cells or more.