Cells from the entire membrane field were counted. All experi ments were repeated at least three times. siRNA transfection MCF 7 DOX cells were transfected with siRNA using Lipofectamine RNAiMAX. The siRNA transfected cells were incubated for 48 h and harvested for Western blot analysis. Reverse transcription polymerase chain reaction Total RNA was isolated from MCF 7 DOX cells using Trizol Bosutinib chemical structure reagent. cDNAs synthesized from 1 ug of total RNA were used as templates in a 50 ul reac tion using the TaqMan RT reagents according to the manufacturers protocol. RT PCR was performed to amplify genes using a cDNA template corresponding to gene specific primer sets. To avoid amplifying genomic DNA, gene primers were chosen from different exons. PCR was performed in a total reaction volume of 25 ul that contained 2 ul of cDNA solution and 0.

2 uM of sense and antisense pri mers. The RT PCR exponential phase was determined on cycles 28 33 to allow quantitative comparisons among the cDNAs amplified from identical reactions. The amplification products were resolved on a 2% agarose gel, stained with ethidium bromide, and visualized on a transilluminator and photographed. Experimental lung metastasis models MCF 7 and MCF 7 DOX cells were injected into the tail vein of Balb c nude mice. Three months after injection, the animals were killed by CO2 inhalation and their lungs were excised. Lung tumor formation was observed and tumor nodules were counted under a dis secting microscope. All animal experiment procedures were approved by the Institutional Animal Care and Use Committee in Korea National Cancer Center.

Gelatin and fibrinogen plasminogen zymography The proteolytic activity of MMP 2, MMP 9, and uPA in CM was analyzed by substrate gel electrophoresis using SDS PAGE gels containing 0. 2% gelatin or 0. 12% fibrinogen and plasminogen. CM from each treatment group was concen trated using an Amicon Ultra 4 centrifugal device and loaded onto gels. After electrophoresis, the gels were washed with 2. 5% Triton X 100 and incubated overnight in zymogram incubation buffer at 37 C. Clear bands indicative of gela tinolytic activity were visualized by staining the gels with Coomassie blue. Gene expression analyses from whole genome Total RNA was isolated and purified from MCF 7 and MCF 7 DOX cells using the TRIzol reagent and RNease Mini kit.

Of those, 500 ng RNA was biotinylated and amplified using the Illumina TotalPrep RNA Amplification Kit according to the manufacturers instructions. The cRNA yield was measured using RiboGreen RNA quantitation kit, and 750 ng of the cRNA sample was hybridized on a human HT 12 expression bead chip for profiling 48,804 tran scripts per sample. Bead Anacetrapib chips were stained with strep tavidin and scanned using an Illumina BeadArray Reader. BeadStudio V3 was used to quantile normalize the data.