MEK-ERK activation is required for expansion of hematopoietic progenitor cells. (A) CD34⁺ cells were starved overnight in the absence of cytokines and in the presence of 0.5% FCS. Cells were left untreated (lanes 1-2) or treated with 5 μM U0126 (lanes 3-4) for 1 hour before stimulation with G-CSF (lanes 2,4) for 15 minutes. Protein lysates were prepared, and Western blot analysis was performed with an antibody against phosphorylated ERK1/2 and phosphorylated p38MAPK, and as a control for equal loading an antibody against total ERK1/2. (B-C) CD34⁺ cells were cultured for 17 days in the presence of G-CSF to induce neutrophil differentiation in the absence or presence of 5 μM U0126. Expansion was determined by (B) counting the trypan blue–negative cells or by (C) performing ³H-thymidine incorporation assays. Data were depicted as a ratio between control and cells treated with U0126. (D) During the 17-day culture period the percentage of apoptotic cells was determined by annexin V staining. Results are presented as means of 4 independent experiments. Error bars represent SEMs.