FIG. 3.

Constitutive expression of miR-K1 attenuates p21-induced cell cycle arrest. U2OS cells were transduced with a control lentiviral vector (pLCE) or a lentiviral vector expressing miR-K1 (LCE/miR-K1). (A) Three days after transduction, cells were cotransfected with RLuc indicator vectors carrying no additional sequences (no target) or 2 perfect matches to miR-K1 or miR-K4, as indicated, and an FLuc internal-control vector. Dual luciferase assays were carried out 24 h later. The RLuc-to-FLuc ratios observed for the miR-K1 and miR-K4 indicator vectors were normalized to those observed for the control vector lacking miRNA targets. The values obtained in cells expressing only EGFP were set at 100%. The error bars are from independently generated cell pools and indicate SD (n = 3). (B) Cells were treated for 24 h with 10 μM Nutlin-3 or DMSO, costained with anti-BrdU-FITC and PI, and analyzed using flow cytometry. S phase, upper gate; G₁, lower left; G₂, lower right. The data shown are representative of two experiments using independently generated cell pools. In experiments 1 and 2, miR-K1-expressing cells arrested ∼2.3-fold and ∼1.9-fold less efficiently than EGFP-expressing cells, respectively. The results from experiment 2 are shown in panel B.