Effect of signaling pathway inhibitors on BMP4- and TPO-induced MK differentiation. Purified CD34⁺ cells were incubated in 4GF serum-free medium in the presence or not of TPO (left panels) or BMP4 (right panels; 50 ng/mL) with or without specific inhibitors of the following signaling pathways: JAK/Stat (AG490, 30 μM), MAPK (PD98059, 2 μM), PI3K (LY294002, 10 μM), and mTOR (rapamycin, 100 nM). (A) After 3 days, cells were harvested and the number of viable cell was evaluated by trypan blue counting. Data represent the percentage of viable cells as a result of the following ratio: (viable cell number/total cell count) × 100. The fold proliferation represents the ratio between the number of viable cells at day 3 and input cells. (B) Cells were placed in functional progenitor assays to determine CFC-MK numbers after 3 days of treatment. Results are expressed as numbers of colonies per 1000 seeded cells. Results (panels A,B) represent the mean plus or minus SEM of 9 experiments. (C) Phenotypic analysis of the early MK specific marker CD41 was performed after 7 days of treatment by flow cytometric analysis using a Facscalibur cell analyzer (BD Biosciences) and gating on viable cells. Results are presented as percentages of positive viable cells plus or minus SEM of 6 experiments. * indicates statistically significant difference from those in nontreated cells (P < .05).