Figure 6.

A: Overexpressed Tel decreases Bcl-xl protein expression. H1299 and I45 cells were seeded into six-well plates (10⁶ cells) and were transfected with Tel and β-galactosidase expression vectors. Seventy-two hours after transfection, Tel and Bcl-xl expression was determined by Western blot. B: Serum starvation enhanced repression by Tel of Bcl-xl transcription. I45 cells were seeded into six-well plates (10⁶ cells) and were transfected with Tel expression vector. The cells were cultured under normal conditions and serum starvation for 48 and 72 hours. Bcl-xl expression was then determined by Western blot. Protein expression was quantified using an UN-SCAN-IT automated digitalized system. C: HGF induces the phosphorylation of Tel in human mesothelioma cells. The mesothelioma cell line, I45, was grown under conditions of serum starvation or serum starvation plus 20 minutes of HGF (100 ng/ml) stimulation. The endogenous Tel proteins were precipitated from cell lysates using their respective antibodies. The immunoprecipitates were then analyzed by Western blot using phosphorylated-Ser/Thr antibodies. The loading was normalized by measuring the Tel expression levels in the cell lysates by Western blotting. D: Increased cytoplasmic localization of Tel in mesothelioma cells after HGF treatment. Twenty-four hours after serum starvation, I45 cells were exposed to HGF stimulation for 20 minutes. The cells were then washed in PBS and fixed with ice-cold acetone and incubated with Tel antibodies followed by a fluorescein isothiocyanate (FITC)-labeled secondary antibody. Fluorescein signals were visualized under an Olympus IX71 fluorescent microscope. E: CHIP analysis of Tel binding to the Bcl-xl promoter. I45 cells were serum starved for 24 hours and then subjected to treatment with/without HGF (100 ng/ml) for 20 minutes. The results of the CHIP assay demonstrated decreased binding of Tel to the Bcl-xl promoter after HGF treatment. DAPI, 4,6-diamidino-2-phenylindole; FBS, fetal bovine serum.