Effect of rapamycin on the expression of different proteins regulating cell cycle. (A) CD34⁺ cells were cultured in the presence of TPO without Rapa or with Rapa (100 nM) added at day 0 and day 3. Cells were analyzed by Western blot at day 6. To control the effect of Rapa, the diminution of p70S6K phosphorylation was ascertained in each experiment. Two cyclins (E and D3) and 2 inhibitors of the cycle (p21 and p27) were studied. The diminution of only cyclin D3 and p21 was detected. (B) Real-time RT-PCR was used to quantify p21 and cyclin D3 mRNA in MKs at day 6 of culture. The relative expression of p21 and cyclin D3 was calculated in comparison to mTOR mRNA, which was stable during Rapa treatment. Results are mean ± SD of triplicate determinations from a representative experiment (n = 4).