Samples were analysed for DNA content using a FACScalibur? instrument (BD Immunocytometry Systems, San Jose, CA). Each experiment was repeated at least three times. Apoptosis: Following treatment by drugs (DSL 25 ��g/ml, Rg3 80 ��g/ml and GEM 2.5 ��g/ml) for 24 h, apoptotic cells in the Vandetanib VEGFR inhibitor population were detected using the AnnexinV-FITC apoptosis Detection KIT I (Pharmingen, San Diego, CA), according to the manufacturer’s instructions. Briefly, cells were washed twice with cold cell staining buffer, and then cells were resuspended in Annexin V binding buffer at a concentration of 1��106 cells/ml. Annexin V-FITC (5 ��l) and 7-AAD viability staining solution (5 ��l) were added in 100 ��l cell suspension in a 5 ml test tube. Cells were gently vortexed and incubated for 15 min at room temperature (25��) in the dark.

400 ��l of Annexin V binding buffer was added to each tube, and cells were analysed by flow cytometry with proper machine settings. Immunohistochemical staining: The immunohistochemical staining (IHC) was performed as per the reported procedure[17]. Briefly, a layer of appropriate size glass coverslips were placed in 24-well plates, and 1��104 cells were seeded in the wells. After treatment by drugs (DSL 25 ��g/ml, Rg 3 80 ��g/ml and GEM 2.5 ��g/ml) for 24 h, the cells were fixed with 4% polyoxymethylene. Following quenching endogenous peroxidase with 3% H2O2 in methanol for 15 min, the cells were incubated for 30 min using block buffer (3% bovine serum albumin (BSA) and 0.1% Triton-100).

The cells were then incubated overnight at 4�� with mouse monoclonal antibodies against human ES, VEGFR-2, caspase-3, caspase-8, caspase-9, dr5, bcl-2 and survivin in 1:200 dilution. The next day, the cells were incubated with secondary antibody for 2 h at room temperature. The bound secondary antibody was visualised by the activity of the horseradish peroxidase conjugate using 3,3-diaminobenzidine tetrahydrochloride (Sigma, UK) as a substrate. The cells were counter-stained with hematoxylin. For semiquantitative evaluation of the IHC by Image-Pro Puls (IPP) 5.1 image analysis software, 20 random visual fields were examined using the LeicaQ550cw image analysis system (Germany). Western blotting: After treatment by drugs (DSL 25 ��g/ml, Rg3 80 ��g/ml, GEM 2.5 ��g/ml) for 24 h, the cells were lysed in the RIPA buffer (50 mM Tris�CCl, pH 7.

4, 150 mM NaCl, 1 mM MgCl2, 0.5% NP-40, 1 mg/ml BSA, 0.1 mM PMSF). The untreated cells were used as controls. Protein concentrations in the cell extracts were determined using the BCA Protein Assay reagents (Pierce Biotechnology, Rockford, USA), according to the manufacturer’s instructions. Samples were separated by SDS-10% Cilengitide polyacrylmide gel electrophoresis, and were electroblotted onto polyvinylidene difluoride membranes.