Specific down-regulation of Notch1 by siRNA induces apoptosis. (A) Down-regulation of Notch1 and Hes expression by Notch1 siRNA. Whole-cell extracts were prepared from Seax cells 24 to 72 hours after transfection with siRNA or scRNA (control) and probed with antibodies against intracellular Notch1 and Hes1, as in Figure 1. Band intensity quantification showed a decrease in Notch1 and Hes expression by 73% and 45%, respectively, 72 hours after transfection. (B) Influence of Notch1 siRNA on cell proliferation. Cells were transfected as in panel A and enumerated by flow cytometry 24, 48, and 72 hours after transfection. Bars represent mean values (n = 3) ± SD. *P < .05 compared with the scRNA-treated, control group. (C) Increase in caspase 3/7 activity in SeAx cells transfected with Notch1 siRNA. Caspases were measured, as described in Figure 2, 24 hours after transfection. Data are mean ± SD. *P < .001. (D) Induction of annexin V-positive, apoptotic cells 24 to 72 hours after transfection with Notch1 siRNA compared with the scRNA-transfected cells. Flow cytometry and enumeration of cells were done as described in Figure 2.