Figure 3

TGF-β1 signaling regulates XIAP gene expression in meningioma cells. (a) Cell lysates from IOMM-Lee and CH157-MN cells that were treated with specified doses of TGF-β1 were subjected to western blotting for the detection of XIAP, SMAD-2, and pSMAD-2 using specific antibodies. GAPDH served as a loading control. Each blot is indicative of three independent experiments. (b) Band intensities from each blot were quantified using ImageJ software and the relative expression was plotted. Columns represent mean±S.D. of three independent experiments. *P<0.05, significant difference from control without treatment. (c) IOMM-Lee and CH157-MN cells were grown to 90% confluence and then stimulated by TGF-β1 (30 ng/ml) in conjunction with anti-TGF-β1 antibody or nonspecific IgG (NsAb) for 48 h (10 μg/ml). Cell lysates were probed for XIAP and pSMAD-2. GAPDH served as a loading control. (d) For invasion assays, cell for 24 h were trypsinized and allowed to invade Matrigel-coated transwell inserts for further 24 h at 37 °C. Then, the cells were fixed and stained. Invaded cells were photographed under a light microscope and percent invaded cells were plotted. Columns: mean; bars: ±S.D.; n=3; ^#P<0.05 versus control. (e) IOMM-Lee and CH157-MN cells grown to 90% confluence were pre-treated with vehicle (mock), PI3K inhibitor (LY294002;10 μM), or MEK1/2 inhibitor (U0126; 10 μM) for 30 min and then stimulated by TGF-β1 (30 ng/ml) as indicated. Cell lysates were probed for XIAP and pSMAD-2 after 48 h of treatment. GAPDH served as a loading control. (f) The band intensities were quantified with ImageJ software, and the relative protein expression was plotted. Each blot represents three independent experiments and each column designates mean±S.D. of three independent experiments. ^#P<0.05, significant difference from TGF-β1 stimulated group of cells