JWA null mutant mice were produced by intercrossing the JWAD2/ mice

Formation Assay Cells growing in log phase 14192894 were seeded at a density of 1000 2000 cells/well into 60 mm dishes in complete medium. After allowing the cells to adhere for 24 h, medium was replaced with complete medium containing ACCA at the indicated concentrations. Cells were allowed to grow for 34 weeks, with a medium change containing ACCA in complete medium, performed every fourth or fifth day. Colonies were then fixed and stained with Methylene blue-50% ethanol and counted. order 1022150-57-7 Individual assays were performed in triplicate with a total of three plates per data point. Apoptosis Analysis by Flow Cytometry Tumor cells were treated with the indicated concentrations of ACCA for 48h a 37uC. Cells were ‘trypsinized and washed twice with phosphate buffered saline and collected by centrifugation at 1000 rpm for 5 minutes at room temperature. Aliquots of cells were resuspended in complete media and then stained with fluorescein isothiocyanate-labeled Annexin-V kit according to the manufacturer’s instructions. PI was added to the samples after staining with Annexin-V kit to exclude late apoptotic and necrotic cells. Flow cytometry was performed immediately after staining. Western Bloy Analysis After treatments, ice-cold PBS solution was used two times to rinse cells. Cells were then lysed with cell lysis buffer and dishes incubated for 1030 min at 4uC. Cells were scraped into lysis buffer, and lysates were clarified by centrifugation. Protein concentrations was determined using a kit from Bio-Rad and western blot analyses were performed as previously described. Aliquots were solubilized in Laemmli buffer, separated by SDSPAGE, and transferred to nitrocellulose membranes. The following antibodies were used: rabbit anti-MCT1, mouse anti-Bax, rabbit anti-Bcl2, and anti-EF-1a. Cell Invasion, Migration and in Vivo Tumorigenicity Assays Transwell invasion and migration assays were performed as described in a modified Boyden chamber. Briefly, the Matrigel was allowed to rehydrate for 2 h at room temperature by adding warm, serumfree DMEM. The wells of the lower chamber were filled with DMEM containing 10% NuSerum, and the chambers were each assembled by placing the uncoated membrane between the lower and upper compartments according to the manufacturer’s instructions. MDA-231 cells were seeded in the upper compartment in serum-free DMEM containg 0.1% BSA. ACCA was then added at the indicated concentrations to the upper chambers and incubated for 48 h at 37uC in a 5% CO2 humified incubator. For migration, cells were added to the upper compartments of the BioCoat chambers supplemented with the ACCA Affects Breast Cancer Cell Growth indicated concentrations of ACCA. The wells of the lower chamber were filled with RPMI 1640 containing 10% NuSerum, and the chambers were each assembled similarly to the method described above 22634634 for migration assay. The migration assay was carried out at 37uC for 1518 h at 37uC. Cells that invaded the Matrigel or migrated to the underside of the coated membrane were fixed, stained with the RAL 555 staining kit. Stained cells were counted and normalized relative to the number of seeded cells. Experiments were assayed in triplicate, and at least 10 fields were counted in each experiment. In vivo tumorigenicity assay was performed as described. Briefly, exponentially growing cells MDA-231 cells were suspended in PBS and mixed in a 1:1 ration with Matrigel. Then, 100 hundred ul of cells were inoculated s.c. on the right flank of each