SCL is expressed in Kit⁺cells and controls Kit-dependent cell survival. (A) Hematopoietic progenitors coexpress Scl and Kit. Scl gene transcription was monitored by β-galactosidase staining (+FDG) of bone marrow cells from Scl^LacZ mice. The substrate was omitted in the negative control (–FDG). (B) Kit⁺ populations enriched in hematopoietic progenitors, CMP, MEP, and GMP, were purified from wild-type (WT) bone marrows. Scl expression was analyzed by quantitative RT-PCR. Messenger (mRNA) levels were normalized using Hprt as an internal control. The means ± standard deviation (SD) of 3 experiments are shown. (C) These progenitors were maintained in culture with SF and IL-11 or GM-CSF and IL-3. Viable cell counts were monitored at the indicated times. (D) SF but not GM-CSF stimulation of Kit⁺Sca⁻Lin⁻ progenitors upregulates Scl mRNA. Purified Kit⁺Sca-1^–Lin^– progenitors were stimulated with SF, GM-CSF, or control medium. Scl expression was determined at different time points by quantitative RT-PCR. mRNA levels were normalized using S16 as an internal control and then compared with expression levels at t = 0. (E) Strategy used to test the role of Scl in the survival of Kit⁺ cells. The 5-fluorouracil (5-FU) mouse bone marrow cells were infected with MSCV-neo^r retrovirus carrying human SCL, a DNA-binding defective mutant (ΔbSCL), or the control empty vector. (F) Kit⁺ cells were analyzed for the presence of human SCL by immunofluorescence staining with a monoclonal mouse anti-human SCL (thick line). Staining with the secondary antibody alone (goat anti-mouse) served as negative control (thin line). (G) After retroviral infection, cells were stimulated with either early acting cytokines (SF + IL-11) or myeloid cytokines (GM-CSF + IL-3) for 2 days in selective media (G418) prior to staining with Kit antibodies and Annexin V (20% to 25% infection efficiency). Dead cells were excluded from analysis by propidium iodide staining. The numbers shown represent the percentages of cells within each quadrant. Data shown (A-G) are typical of 2 or 3 independent experiments.