5-mm diameter, 8-μm pore size polycarbonate membrane). In the upper chamber 1 × 105 cells Epoxomicin molecular weight in 0.2 mL of serum-free medium were placed, while in the lower chamber medium containing 25 μg/ml fibronectin was loaded. Having migrated to the lower surface of filters, the cells were stained with hematoxylin solution. After

6 h for the second incubation, five fields in each well were counted for number of cells. Three wells were examined for each condition and cell type, and the experiment was also repeated for three times. The cell invasion assay was conducted by using 100 ml/well matrigel-precoated 24-well invasion chambers, with learn more filters coated by extracellular matrix on the upper surface. Five fields in each well were counted after incubation for 16 h. Assay of tumorigenicity Fourteen of 5 to 6-week-old female BALB/c mice were divided into two groups (seven mice per group) and inoculated subcutaneously

with 200 μL of Eahy926 cell and A549 cell suspension (5 × 107/ml) respectively. The growth of tumor was observed regularly. After two weeks, the mass of tumor inoculated, the liver and the lungs of mice were taken, fixed in 40 g/L formaldehyde, and cut into sections. Finally, slices ARN-509 of these specimens were stained with regular HE method and observed under microscope. Two-dimensional electrophoresis Eahy926 and A549 cells (2 × 107/ml) were solubilized in 1 ml of cell lysis solution (8 M urea, 4% CHAPS, 2 mmol/L TBP, 0.2% ampholyte, traces of bromophenol blue) on 4°C for 20 min. Insoluble material was removed by centrifugation at 15000 rpm at 4°C for 30 min. Protein concentration was determined by the method of Bradford. Samples were frozen at -70°C, and thawed immediately

before use. For 17 cm IPG Ready Strips, 1 mg of protein was loaded. After rehydrating for 14 h, isoelectric focusing (IEF) was carried out for 1 h at 200 V, 1 h at 500 V and 1 h at 1000 V continuously; then a gradient was applied from 1000 to 8000 for 1 h and finally at 8000 V for 8 h to reach a total of 72 KVh at 20°C. Following IEF separation, gel strips were incubated in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS) with 10 mg/mL DTT for 15 min, followed in equilibration buffer with 25 mg/mL iodoacetamide for 15 min. Then strips were loaded on 12.5% SDS-PAGE gels, and electrophoresised for 20 min at a constant current Arachidonate 15-lipoxygenase of 10 mA and then at 30 mA per gel until the bromophenol blue reached the bottom of the gels. Subsequently, the gels were stained with CBB R-250, and destained with 40% methanol, then with 10% acetic acid. The experiment was replicated for five times. Image analysis and statistical analysis of 2-DE gel The 12 gels were scanned with the Images Scanner GS800 (BioRad) at 300 dpi resolution. Spot detection, quantification, and the analyses of 2-D protein patterns were done with the PDQuest software (version 7.2, BioRad). Then the report of quantitative differences between two gel images was generated.