Effective targeting of the miR-29b and Dnmts in the dKI AML. Whole spleen cells isolated from secondary transplants of leukemic Ly5.2⁺ dKI mice were intravenously injected into sublethally irradiated Ly5.1⁺ syngeneic C57Bl/6 mice. Upon development of AML, Ly5.1 negative selection was performed on bone marrow cells to achieve >95% purity of the Ly5.2 AML blast population. Blasts were then cultured ex vivo in the presence of vehicle or bortezomib up to 24 hours. Each experiment was performed 3 or 4 times as independent replicates. (A) Real-time RT-PCR quantification of pri-miR-29b-2 after 12 and 24 hours of culture in phosphate-buffered saline (vehicle) or various concentrations of bortezomib as indicated in the key. (B) Representative immunoblot of Dnmt1, Dnmt3a, and Dnmt3b on whole cell lysates collected after 24 hours of treatment with vehicle (0), 3, 6.5, 10, or 30 nM bortezomib. Graph shows quantification of protein/actin ratios between vehicle and bortezomib-treated cells calculated from 3 independent experiments. (C) Real-time RT-PCR quantification of Id4 gene expression in murine AML. A decrease in the ΔCT for Id4 was calculated using β-2 microglobulin as an internal control and signifies an increase in Id4 gene expression following 24 hours of culture in 30 nM of bortezomib. (D) Changes in cell growth measured by MTS assays. (E) Changes in apoptosis measured by Annexin V and 7-AAD staining and flow cytometry. (D-E) P values indicate significance of each treatment group compared with vehicle.