Nd ECL immunoblotting detection reagents from Amersham Biosciences (Amersham Life Science, Amersham, UK), and anti-goat IgGHPRO peroxidase linked antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Complete miniEDTA-free protease inhibitor cocktail tablets and Annexin-V-Fluos were purchased from Roche GmbH (Mannheim, Germany). All other reagents were from Sigma Chemical Company (Taufkirchen, Germany). STI571 was kindly provided by Novartis Pharma AG (Basel, Switzerland).Cell culture and MTT assay Human pancreatic cancer cell lines were PNPP site routinely grown in DMEM (Colo-357, and Mia-PaCa-2) or RPMI (Aspc-1, BxPc-3, Capan-1, and T3M4) supplemented with 10 FBS, 100 U/ml penicillin, and 100 /ml streptomycin (complete medium). To assess cell proliferation, the MTT test was employed. Briefly, cells were seeded at a density of 5000 cells/well in 96-well plates, grown overnight and exposed to STI571 alone or in combination with growth factors. After 48 or 72 hours of incubation, 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) was added (50 /well) for 4 hours. Formazan products were solubilized with acidic isopropanol, and the optical density was measured at 570 nm. To determine the GI50 of STI571 (the concentration that causes 50 growth inhibition), graded concentrations of STI571 were added to triplicate wells and GI50 was calculated using 100 ?(T-T0)/(CT0) = 50. T is the optical density of the test well after a 48hour period of exposure to STI571, T0 is the optical density at time zero, and C is the control optical density after 48 hours. All experiments were performed in triplicate. FACS analysis of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28499442 cell death and cell cycle 105 pancreatic tumor cells were seeded into 6-well plates in 1 FBS-containing medium, allowed to adhere overnight and then treated with corresponding GI50 concentrations of STI571. To analyze cell cycle distribution, cells were collected after 48 hours of incubation, washed with PBS and resuspended in 0.5 ml of hypotonic PI buffer (5 /ml propidium iodide, 0.1 Triton X100 and 0.1 sodium citrate), stored overnight at 4 and then analyzed by flow cytometry using BD-LSR (Becton Dickinson and Company, New York, USA). The resulting DNA histograms were interpreted using the Cell Quest Pro software (Becton Dickinson and Company, New York, USA). To determine the degree of cell death, cells were collectedMiaPaCa2T3MSTI571+EGF control STI571+EGF control STI571 STIEGFEGFPEGFRp42 MAPKFigure STI571 phosphorylation on growth factor-induced EGFR Effect of5 Effect of STI571 on growth factor-induced EGFR phosphorylation. Mia-PaCa-2 and T3M4 cell lines were cultured in 1 FCS medium overnight, then treated with 10 ng/ml of EGF and GI50 concentrations of STI571 for 5 minutes. Phosphorylation of EGFR was determined by Western blot analysis with a phospho-EGFR-specific antibody. Equal loading was determined by stripping the membranes and blotting with an antibody to ERK2 (p42). The figure is representative of three independent experiments.after 12, 24 and 48 hours of exposure to GI50 of STI571 and were washed and stained with Annexin-V-FITC (apoptotic death) or PI (necrotic death) according to the manufacturer’s instructions (Roche, Mannheim, Germany).Western blot analysis Cell culture monolayers were washed twice with ice-cold PBS and lysed with buffer containing Tris-HCl (50 mM, pH 7.4), NP-40 (1 ), Na-deoxycholate (0.25 ), NaCl (150 mM), EDTA (1 mM), PMSF (1 mM), Na3VO4 (1 mM), NaF (1 mM) and one t.
