Physiological effects of TFAP2B silencing in aRMS cells. (a) mRNA level of TFAP2B after RNAi-mediated downregulation in Rh4 cells as measured by qRT–PCR analysis. The ratio of siRNA to scRNA (control) treatment is shown. (b) Protein levels of TFAP2B as determined by western blot with 10 μg of nuclear protein from TFAP2B specific – (si) and control siRNA (sc) treated cells extracted 24 and 48 h after transfection. In the positive control lane, mTFAP2B transiently overexpressed in 293T cells is shown as size control. FKHR protein levels were used as loading control. (c) Cell proliferation as measured by MTT assay after different time periods of treatment with TFAP2B-specific siRNA, scRNA and transfection reagent alone (GeneEraser). The means±s.d. (error bars) from three independent triplicate experiments are shown. (d) Apoptosis rate in Rh4 cells transfected with the indicated siRNA as detected by a caspase-3/7 assay at the indicated time points. Mean values of one representative experiment performed in triplicates are shown. (e) Histogram plots of FACS results of cells stained with PI 24 h after treatment with the indicated siRNA. Percentages of PI-positive cells are depicted above the horizontal bars. aRMS, alveolar RMS; qRT–PCR, quantitative reverse transcription-PCR; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI, propidium iodide; RMS, rhabdomyosarcoma; scRNA, scrambled siRNA; siRNA, small interfering RNA.