Figure 7

The pRb pathway mediated miR-383-induced phenotype in NT2 cells. (a) Cells were treated as in Figure 4i. For western blotting analysis of the indicated proteins, cells were harvested 2 h after serum stimulation of growth-arrested cells. (b) The efficacy of siRNAs for cyclin D1, CDK2 and p21 was evaluated by western blot. (c) Cyclin D1 partly rescued NT2 cells from growth inhibition by miR-383. Twenty-four hours after transfection with 100 nM miR-383 mimic, cells were further transfected with 0.1 μg cyclin D1 expression vector (pcDNA3.1/cyclin D1) for 24 h and then subjected to cell viability assay. (d and e) Effects of knockdown of cyclin D1 or p21 on miR-383-induced G1-phase arrest (panel d) and apoptosis (panel e). NT2 cells were cotransfected with the miR-383 mimic and the indicated siRNAs. (f) Immunoblot analysis of phosphorylation of pRb using an antibody specific for phosphorylated Ser²⁴⁹/Thr²⁵² (P-pRb). Cells were treated as in Figure 4i. For western blotting analysis, cells were harvested 24 h after serum stimulation of growth-arrested cells. (g and h) The protein level of CDK4 was determined by western blotting 24 h after transfection with the indicated oligonucleotides (panel g) or cotreatment of the miR-383 mimic with 20 μM of MG-132 or DMSO (panel h). SFM, serum-free medium. Error bars indicate S.E.M. (n=3). ^*P<0.05, ^**P<0.01, compared with negative controls