Validation of the microarray analysis. (A) Quantitative RT-PCR of MCL-1, CXXC6 (B) and CDK6 (C) after 24 hours of transfection with synthetic miR-29a, -29b, or scrambled oligonucleotides in K562 cells. The results are shown as average mRNA expression after normalization with 18s and 2ΔC_t calculations. Data represent the average of 3 independent experiments ± SD. (D) Western blotting of Mcl-1 and Cdk6 protein expression in K562 and Kasumi-1 cells (E) after 48 hours of transfection with synthetic miR-29a, -29b, or controls (scrambled oligonucleotides [SCR] and miR-212) or lentivirus miR-29a, -29b, or empty vector (EV) for Kasumi-1 cells. The protein loading control was performed using GAPDH. (F) Rb phosphorylation at Ser⁷⁸⁰ after 24 hours of transfection with synthetic miR-29b or control (SCR). (G) Quantitative RT-PCR of MCL-1, CXXC6, and CDK6 in 3 primary AML blasts after transfection with synthetic miR-29b or controls (SCR). The results are shown as average mRNA expression after normalization with 18s and 2ΔC_t calculations. Bars represent mean ± SEM. (H) Western blotting of Mcl-1 in 3 newly diagnosed AML patients after transfection with synthetic miR-29b or controls (SCR). Loading control was performed using GAPDH. Mcl-1/GAPDH ratios are shown after densitometry evaluation of the gels. (I) Normalized Mcl-1 protein expression averages relative the mock-transfected primary AML samples. Bars represent SD.