Inhibition of Notch pathway by Notch siRNA or GSI inhibited Rink-1 cell growth.

A, Left panel, Inhibition of Rink-1 cell growth by Notch 1–4 siRNA tested by MTT assay. The results were plotted as means ± SD of three separate experiments having six determinations per experiment for each experimental condition. Middle and Right panel: L-685,458 and DAPT were γ-secretase inhibitors (GSI), which prevent the cleavage of the Notch receptor, blocking Notch signal transduction. GSI significantly inhibited Rink-1 cell growth. Cells were seeded in 96-well plates at 5,000 cells per well and treated with GSI for 72 hours. After treatment, cell densities were determined by MTT assay. Each value represents the mean ± SD (n = 6) of three independent experiments. *P<0.05, compared to the control. B, The expression of Notch pathway was down-regulated in Rink-1 cells treated with GSI or transfected with Notch 1–4 siRNA as assessed by western blotting analysis. C, The expression of Notch target genes was down-regulated in Rink-1 cells treated with GSI or transfected with Notch-2 siRNA or Notch-4 siRNA as assessed by as assessed by real-time RT-PCR.