miRNA-15a and -16 modulates proliferation and cell cycle of MM cells. (A,B) MM cells (pre-miRNA-15a–, -16-1 probe–, and control probe–transfected and nontransfected MM.1S) were harvested at 24, 48, and 72 hours after transfection; DNA synthesis and cytotoxicity were assessed by thymidine uptake and MTT assays, respectively. Nontransfected MM.1S cells were used as controls. P values are indicated. (C) Cells were first arrested and synchronized in G2/M phase by growth in 80-nM nocodazole for 16 hours. Cells were then washed and regrown using fresh media. After 6 hours, cell cycle analysis was performed by propidium iodide staining. (D) MM cells (pre-miRNA-15a–, -16-1 probe–, control probe–transfected) were harvested at 24 hours after transfection. Whole cell lysates were subjected to Western blotting using anti–cyclin D1, –cyclin D3, cdk6, –p-Rb, -Bcl2, and -actin antibodies; nontransfected MM.1S cells were used as controls (ctrl). (E) MM cells (pre-miRNA-15a–, -16-1 probe–, control probe–transfected) were harvested at 24 hours after transfection. Whole cell lysates were subjected to Western blotting using anti–phospho(p)-AKT, -AKT1, -AKT3, –p-ERK, -ERK, p-S6R, and -actin antibodies; nontransfected MM.1S cells were used as controls (ctrl).