miR-28 and closely related miR-151 and miR-708 inhibit Tpo-dependent proliferation of Mo7e cells and target mRNAs coding for proteins involved in proliferation and apoptosis. (A) Mo7e cell lines transduced with a bicistronic retrovirus (pMegix) expressing the GFP protein along with indicated microRNAs. Western blot analyses for MPL and ß-actin (ACTB) protein levels were performed. Normalized MPL protein levels are indicated under the blots. (B) Mo7e cell lines expressing miR-28, miR-708 or miR-151 were grown in the presence of indicated amounts of Tpo or GM-CSF (granulocyte macrophage–colony-stimulating factor) for 4 days. Cell proliferation is represented as fold increase compared with cell lines grown without cytokines. Shown are averages of triplicates ± SD of 1 representative experiment of 3. (C-D) 3′UTRs () of indicated mRNAs or 3′UTRs mutated for miR-28 target sites (■) were cloned after the stop codon of the renilia luciferase in the psi-CHECK-2 reporter vector. These luciferase reporter vectors were cotransfected with miR expressing vectors. (E-F) CD34+ hematopoietic progenitors were infected with pMegix bicistronic retrovirused coexpressing miR-28 with the GFP or the control retrovirus expressing only the GFP. After 14 days of culture, proplatelet-bearing CD41/GFP-positive megakaryocytes were counted. Shown in panel E are numbers of proplatelet-bearing CD41/GFP positive megakaryocytes (average of triplicates + SD of 1 representative experiment).