Differential expression of MEK1 target genes. (A) CD34⁺ hematopoietic progenitor cells, transduced with MEK1:ER*, were cultured in the presence of G-CSF to induce neutrophil differentiation. Two days after transduction, eGFP-positive cells were separated from the nontransduced cells by FACS and resuspended in culture medium containing G-CSF and IL-3. After 6 days of differentiation, cells were washed twice with PBS and starved overnight in the absence of cytokines and in the presence of 0.5% FCS. Subsequently, cells were incubated with 0 or 20 nM 4-OHT for 12 hours; supernatants were collected; and IL-8, CCL2, and CCL3 protein expression was determined by using Luminex multiplex analysis as described in “Analysis of differential gene expression in response to MEK1 activation.” Results are presented as the means of 2 or 3 independent experiments. Error bars represent SEMs. (B) Protein lysates were prepared from CD34⁺ cells transduced with MEK:ER* and treated with solvent, 20 or 100 nM of 4-OHT overnight. Western blot analysis was performed with an antibody against p21Cip1, phosphorylated ERK1/2, or tubulin as a control for equal loading. (C) CD34⁺ cells, either transduced with MEK1:ER* or left untreated, were differentiated toward neutrophils in the presence of G-CSF. After 6 days of culture, cells ectopically expressing MEK1:ER* were washed and resuspended in culture medium either in the presence or absence of 4-OHT to prepare conditioned medium. After 7 days of culture, untreated control granulocyte progenitors from the same donor were resuspended in either conditioned medium or culture medium containing 4-OHT as a control. Proliferation over a 3-day period was determined by cell counting. (D) CD34⁺ cells, either transduced with MEK1:ER* or left untreated, were cultured in the presence of G-CSF to induce neutrophil differentiation. After 6 days of culture, cells ectopically expressing MEK1ER were washed and resuspended in culture medium either in the presence or in the absence of 4-OHT. A transwell migration was performed with untreated control granulocyte progenitors from the same donor in response to conditioned MEK1ER medium or as a control culture medium with or without 4-OHT. The assays were performed for 4.5 hours, after which the percentage of migration was determined by FACS analysis. Data were expressed as an average of 4 individual experiments. Error bars represent SEMs.