Conditional activation of MEK1 promotes expansion of myeloid progenitors. (A) Protein lysates were prepared from CD34⁺ cells transduced with MEK:ER* and treated with solvent, 20 or 100 nM of 4-OHT for 30 minutes. Western blot analysis was performed with an antibody against phosphorylated ERK1/2 or tubulin as a control for equal loading. Similar results were obtained in 4 independent experiments. (B) CD34⁺ cells were transduced with MEK1:ER*, sorted by FACS from the nontransduced cells, and differentiated to neutrophils in the presence or absence of 20 nM 4-OHT. Expansion was determined by counting the trypan blue–negative cells. (C) CD34⁺ cells were transduced with MEK1:ER*, sorted by FACS from the nontransduced cells, and differentiated to neutrophils in the presence or absence of 20 nM 4-OHT. During the 17-day culture period the percentage of apoptotic cells was determined by annexin V staining. (D) CD34⁺ cells, transduced with MEK:ER and sorted by FACS, were seeded in 60-well plates at a density of 1 cell per well in normal culture medium containing G-CSF in the presence or absence of 20 nM 4-OHT. After 12 days, wells with colonies were scored. (E) CD34⁺ progenitor cells transduced with MEK1:ER* were differentiated toward neutrophils in the presence or absence of 4-OHT. After either 3 and 7 days of culture, cells were plated in CFU assays in the absence of 4-OHT, and colony formation was analyzed after 12 days of culture. Results are presented as means of 4 independent experiments. Error bars represent SEMs.