In this study, M13SV1R2N1 cells were cultured in the MSU 1 medium supplemented with growth factors/hormones and 5% fetal bovine serum. After one week culture in this medium, M13SV1R2N1 cells were subcul tured in the basic MSU 1 medium with 5% FBS without other growth factors/hormones below and passaged more than 10 times. Immunocytochemical analysis of gene expression For immunostaining, cells were fixed by 4% paraformal dehyde in phosphate buffered saline. After rinsing with PBS, Inhibitors,Modulators,Libraries the cells were permeabilized for 10 min. These cells were then incubated with primary antibodies at 25�� overnight. The following day, these cells were incubated with a secondary antibody conjugated Inhibitors,Modulators,Libraries with fluorescein isothiocyanate for 1 hr at 25��. For nuclear staining, the cells were washed with PBS before incubation with 4, 6 diamidino 2 phenylin dole for 5 min.

Inhibitors,Modulators,Libraries Flow cytometric analysis of gene expression Following trypsinization, cells were strained through a 40 uM nylon mesh to ensure the obtaining of single cells and suspended in ice cold solution for a density Antibodies were added to the cell suspension at concen trations suggested by the manufacturer and cells were incubated at 4 C in the dark for 45 min. Then the cells were incubated with a secondary antibody conjugated with FITC or PE for 1 hr at 4 C. These labeled cells were washed twice, suspended in PBS and analyzed using a flow cytometer. As negative controls, cells were stained with either isotype matched control antibodies or with no primary antibody. No difference was observed between these two controls.

Western blotting The proteins were extracted with 20% SDS lysis solution containing several protease and phosphatase inhibitors. Protein concen trations were measured using Biorad Protein Quantifica tion kit. Equal amounts of protein were separated by 12% SDS PAGE and transferred from the gel to PVDF membranes. Immunoblotting Inhibitors,Modulators,Libraries was carried out using monoclonal antibody. This was then followed by incubation with horseradish peroxidase conjugated secondary antibody and detected with the ECL chemilu minescent detection reagent. The membranes were exposed to X ray film for 15 s to 3 min. Reverse transcription polymerase chain reaction 5 ug of total RNA extracted from cells were used to synthesize the first strand cDNA, using the Reverse Transcription System according to the manufacturers protocol.

PCR amplification was car ried out by using 1 uL of the first strand cDNA as a template in a total volume of 15 uL containing 1 uL of each primer and 7. 5 uL of EconoTaq PLUS GREEN Master Mix Kit minute denaturation Inhibitors,Modulators,Libraries at 95 C, the reactions were cycled 30 times with 45 seconds denaturation at 95 C and 30 seconds annealing at 55 C and then exten sion at 72 C for 1 min. The reactions were performed in the DNA Thermal Cycler 480. The last poly merization step was at 72 C for 10 min. Invasion assay Cells were inoculated into 24 well Matrigel selleck chemicals DAPT secretase Invasion inserts.