siRNA design The design of 19 nucleotide target sequences were based on a computer algorithm and 5′-GCCACCGCTGCGGCCTTCTTC-3′ was selected as the target sequence. These were separated by a nine-nucleotide noncomplementary spacer (5′-TTCAAGAGA-3′) from the reverse complement of the same 19-nucleotide sequence. For preparation of recombinant plasmids, oligonucleotides (64 bp) were ligated into the mammalian expression vector, pSilencer 3.1-H1 neo (Applied

DNA Damage inhibitor Biosytems Japan, Tokyo, JAPAN) at the BamHI and HindIII cloning sites. Recombinant MUC5AC-pSUPER gfp-neo constructs were used to transform Escherichia coli DH5, which were selected on ampicillin-agarose plates and verified by sequencing. Cell proliferation assay Cell proliferation was determined by the 3H-thymidine uptake assay. After 24 h or 48 h of incubation, radioactivity was measured using cell harvester and counters. Experiments were performed in triplicate, and values are expressed as cpm/well. Adhesion assay The adhesion assay was done as described before [14]. Briefly, A 96-well microtiter plate was coated with Matrigel (2 μg/well), laminin

(4 μg/well) and fibronectin (4 μg/well). Cancer cells (4 × 105) were then seeded onto these components. No chemicals signaling pathway for extracellular stimulation

were added. Cells were allowed to adhere O-methylated flavonoid to each well for 30 min at 37°C and were then gently washed three times with PBS. The adhesive cancer cells to extracellular components were evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide colorimetric assay (MTT assay). The percentage of cells adhering was calculated as follows: % binding = (absorbance of treated surface – ECM component)/absorbance of total surface × 100. All experiments were performed in triplicate. Invasion assay Invasion activity of cancer cells was measured by the method of Albini et al. [15] with some modifications. Briefly, cancer cells (1 × 104/ml, 200 μl) were seeded in the upper chamber separated with a 12-μm membrane filter coated with 50 μg of Matrigel without adding extracellular stimuli. After incubation for 72 h at 37°C, cancer cells invading the lower chamber were manually counted under a microscope. Six find more randomly selected fields were counted for each assay. Mean values from six fields were calculated as sample values. For each group, culture was performed in triplicate. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) Total RNA extraction and cDNA amplification were as described previously [16].