Figure 6.

miR-16 regulates the cell cycle. (A) Control overexpression or miR-16 overexpression oligonucleotides were transfected in HeLa cells (50 nM final concentration). The next day, cells were reseeded and at given time points the number of cells was counted. (B) Control overexpression or miR-16 overexpression oligonucleotides were transfected in HeLa cells (50 nM final concentration). After 24 h, cells were reseeded and the next day harvested and used for cell-cycle analysis by FACS. (C) Cell-cycle analysis using HeLa cells transfected twice either with control knockdown or miR-16-specific knockdown oligonucleotides (50 nM final concentration) and 24 h later reseeded. After 32 h, cells were treated with UVC and 24 h later harvested for cell-cycle analysis by FACS. (D) The full-length cyclin D1 3′UTR was cloned downstream a Renilla luciferase (RLuc) gene (Psi-CHECK2 vector). HEK293T cells were transiently transfected with either the RLuc–cyclin D1-3′UTR vector or RLuc with a control 3′UTR in combination with a specific miR-16 (50 nM) or control (50 nM) oligonucleotide. A ubiquitously expressed firefly luciferase gene, which is also present in the vector, was used as a transfection control. Luciferase activity was measured 24 h after transfection. (E) The full-length cyclin E1 3′UTR was cloned downstream a Renilla luciferase (RLuc) gene (Psi-CHECK2 vector). HEK293T cells were transiently transfected with either the RLuc–cyclin E1-3′UTR vector or RLuc with a control 3′UTR in combination with a specific miR-16 (50 nM) or control (50 nM) oligonucleotide. A ubiquitously expressed firefly luciferase gene, which is also present in the vector, was used as a transfection control. Luciferase activity was measured 24 h after transfection.