miR33b directly targets PIM-1. (A) Sequence alignment of the miR33b seed sequence with pim1 3′-UTR. Matched nuclear acid base pairs were labeled in red and deletions are labeled as “−.” (B). 293T cells were transiently cotransfected with reporter plasmids (pmiR-vector, pmiR-PIM1-WT, and pmiR-PIM1-MT), miR (miR33b or control miR), and Renilla luciferin with Lipofectamine 2000, and cells were harvested to measure the fluorescence of firefly and Renilla luciferin accordingly. (C) MM.1S cells were transiently cotransfected with reporter plasmids (pmiR-vector, pmiR-PIM1-WT, and pmiR-PIM1-MT), miR (miR33b or control miR), and Renilla luciferin using the Cell Line Nucleofector Kit V. Cells were harvested and lysed 10 hours after transfection, followed by measurement of relative fluorescence intensity of firefly and Renilla luciferin. (D) MM.1S cells were cotransfected with reporter plasmids (pmiR-vector, pmiR-PIM1-WT, and pmiR-PIM1-MT) and Renilla luciferin and then treated with MLN2238 (12nM). Cells were lysed and subjected to luciferase reporter assay using the Dual-Luciferase Reporter Assay System. Luciferase activities were analyzed as the relative activity of firefly to Renilla. Results shown are means ± SD (n = 3). ***P ≤ .001.