Figure 4

Silencing of endogenous IRF1 enhances miR-383-mediated effects on cell cycle and apoptosis in NT2 cells. (a) The efficacy of IRF1 siRNA was evaluated by western blotting after 48 h transfection with either siRNA negative control (si.NC) or IRF1 siRNA (si-IRF1). (b–d) Knockdown of IRF1 inhibited cell proliferation (panel b), induced G1-phase arrest (panel c) and activated apoptosis (panel d). Cell viability (panel b) was measured by the cell counting kit-8 assay after transfection with 5, 10, 20, 30, 50, 100 nM si.NC or si-IRF1. Proportions of cells in the G1, S, and G2/M phases of the cell cycle (panel c) and apoptotic rates (panel d) were determined using flow cytometry. (e–g) Silencing of IRF1 enhanced miR-383-induced proliferation suppression (panel e), G1-phase arrest (panel f) and apoptosis (panel g). Cell viability, cell-cycle profiles and apoptotic rates were analyzed after cotransfection with 100 nM miR-383 mimic and 30 nM si-IRF1 into NT2 cells. (h) IRF1 was induced rapidly by serum. NT2 cells were growth arrested by serum starvation for 24 h and then harvested for western blot analysis at the indicated time points after serum stimulation. Untreated cells were also included as a control (Exp). (i) Overexpression of miR-383 or silencing of IRF1 inhibited serum-inducible IRF1 protein expression. Cells were transfected with the indicated oligonucleotides after serum starvation for 6 h and growth arrested for a further 18 h. For western blot analysis, cells were harvested 2 h after serum stimulation. SFM, serum-free medium. Error bars indicate S.E.M. (n=3). ^*P<0.05, ^**P<0.01, ^***P<0.001, compared with negative controls