Figure 7

Effects of γ-secretase inhibitor (GSI) on proliferation, and invasion of SAS cells. (a) SAS cells were incubated with various concentration of GSI or dimethyl sulfoxide (DMSO) for the indicated periods and then examined for viability via a WST assay. GSI significantly inhibited cell proliferation in a dose-dependent manner. The Y axis shows the changes in absorbance at 450 nm; bars: s.d. of triplicate samples. *P<0.05; **P<0.01, analyzed by Student’s t-test. The photomicrographs show representative images of SAS cells under each condition, as described below. Lysates from SAS cells incubated with 1% DMSO or 10 μM GSI for 48 h were immunoblotted using antibodies as indicated. Decreased expressions of Notch1 and intracellular Notch domain (NICD), phosphorylated histone H3 (p-H3) and phosphorylated Akt (p-Akt) were confirmed. (b) Western blotting of Notch1 signaling-related proteins 48 h after GSI treatment. Note the decreased expressions of Notch1, NICD, p-H3 and phosphorylated AKT (p-AKT) in the GSI-treated cells. The expression of β-actin was used as an internal control. The experiment was performed in triplicate. Images on microscope of SAS cells under each condition. Original magnification, × 100. (c) The effects of GSI on TNF-α-dependent SAS cell invasion. SAS cells were incubated with DMSO or GSI for 24 h in the presence of TNF-α. Cells that penetrated the Matrigel-coated membrane were fixed and stained. The photos are representative fields of invasive cells on the membranes. The numbers of invaded cells obtained in three independent experiments were counted and statistically analyzed. The number of invaded cells enhanced by TNF-α was significantly decreased under the GSI treatment conditions. The bar graphs represent the average numbers of cells on the underside of the membranes. The Y axis shows the numbers of cells invading the underside of the membranes. Mean±s.d. of three independent experiments. *P<0.05; **P<0.01, analyzed by Student’s t-test. The photos are representative fields of invasive cells on the membranes. Original magnification, × 100. Hes, hairly enhancer of split; OD, optical density.