Ultrastructure and protein-expression levels of GATA-1 and C-MPL in rapamycin-treated versus control CB-MKs. (A) Representative electron photomicrograph of a CB-MK cultured in 0.01% DMSO (controls) exhibiting a large cytoplasm, a large cytoplasm/nucleus (Nu) ratio, abundant granules (Gr), and an open DMS. (B) Ultrastructural characteristics of MKs treated with rapamycin showing a striking reduction in cytoplasm, nearly absent granules, and a lack of DMS. Features of very immature MKs were frequently observed, including multivesicular bodies (MVB) and dense compartments (DC). (C) Under the same experimental setting described in Figure 4, MKs were starved overnight and then stimulated with TPO in the presence or absence of rapamycin (50nM). Pretreatment with rapamycin significantly suppressed phosphorylation of S6K and 4E-BP-1 upon TPO stimulation in CB-MKs. (D) In separate experiments, MKs treated with rapamycin from day 11 until day 14 of culture had significantly lower protein levels of GATA-1 and C-MPL compared with control MKs. Bars reflect the means ± SEM of 3 independent experiments. *P < .05.