PKB mediates lineage choice decisions via regulation of C/EBPα. (A) CD34⁺ cells were retrovirally transduced with GSK-3CA or eGFP alone. Retrovirally transduced CD34⁺ cells were cultured in presence of either (A) G-CSF to induce neutrophil development or (B) IL-5 to induce eosinophil differentiation After 17 days of culture, transduced cells were separated from the nontransduced cells by FACS, and cytospins were made. Cytospins were stained with May-Grunwald Giemsa solution. (A-B) Data were expressed as the percentage of differentiated neutrophils or eosinophils. (C) CD34⁺ cells were retrovirally transduced with C/EBPαWT, C/EBPαT222/226A, or eGFP alone. Retrovirally transduced CD34⁺ cells were cultured in presence of either IL-5 to induce eosinophil differentiation or G-CSF to induce neutrophil development. After 17 days of culture, transduced cells were separated from the nontransduced cells by FACS, and cytospins were made. Cytospins were stained with May-Grunwald Giemsa solution. (D) Data were expressed as the percentage of differentiated neutrophils. (E) Data were expressed as the percentage of differentiated neutrophils that developed during the 17-day culture period in presence of IL-3 and IL-5. (F) Data were expressed as the percentage of differentiated eosinophils. (G)A schematic model showing the effect of regulation of PKB activity on regulation of myelopoiesis. Activation of PKB results in inhibitory phosphorylation of GSK-3. Active GSK-3 phosphorylates and thereby inhibits activation of C/EBPα. Unphosphorylated, active C/EBPα inhibits expression of JunB. Both activation of PKB and C/EBPα induces neutrophil differentiation, whereas eosinophil development is inhibited. Inhibition of PKB results in activation of GSK-3, phosphorylation of C/EBPα, and induction of eosinophil differentiation at the expense of neutrophil development. Error bars represent SEM.