Effect of rapamycin on cell size, differentiation, and proplatelet formation of megakaryocytes. CD34⁺ cells were cultured in the presence of TPO and Rapa (100 nM) added at different days of culture (TPO + Rapa) or without Rapa (TPO). (A) Effect of Rapa on cell size. MKs were analyzed by flow cytometry, the mean forward scatter height was used as a parameter of cell size and was measured for each ploidy level (from 2N to 16N) determined by propidium iodide staining of MKs. (i) Rapa was added to the culture at day 0, day 3 and day 6. Only MKs expressing CD41 were analyzed on day 9. (ii) Rapa was added to the culture at day 6. Only MKs expressing CD41 were analyzed on day 9. (iii) Rapa was added to the culture at days 0, 3, and 6. Only MKs expressing von Willebrand factor (VWF) were analyzed on day 9. (B) Effect of Rapa on MK differentiation. Rapa was added to the culture at days 0, 3, 6, and 9. On day 6 (D6) and 12 (D12), the percentage of mature MKs expressing CD41 and CD42 antigens was analyzed by flow cytometry. (C) Effect of Rapa on the protein level of 3 transcription factors (GATA-1, FLI-1, and TAL-1) regulating megakaryocytopoiesis. Rapa was added to the MK culture at day 0 and day 3. Western blot analyses were performed at day 6 on nuclear (N) and cytoplasmic (C) protein extracts from total cultures treated or not with Rapa. 293 cells were used as a negative control for GATA-1, FLI-1, and TAL-1 expression and HDAC-1 was used as an internal control of nuclear protein integrity. (D) Effect of Rapa on mRNA level of GATA-1, FLI-1, and TAL-1 was evaluated by real-time RT-PCR at day 6 of culture in the presence or absence of Rapa. The relative expression of these transcription factors was calculated in comparison to mTOR mRNA, which was stable during Rapa treatment. (E) Rapa was added to the culture only at day 9 (D9; left histogram) or at days 0, 3, 6, and 9 (D0, 3, 6, 9; right histogram). At day 9, the cells were seeded at 2 × 10³ cell/well in 96-well plate. At day 12, the percentage of MKs forming proplatelets was estimated by counting MKs exhibiting one or more cytoplasmic processes with areas of constriction among 500 cells. Each figure shows one representative analysis of 3 repeated experiments with similar results. Results in panels C and E are the mean ± SD of triplicate determinations from a representative experiment (n = 2).