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Cl66 cell monolayers were trypsinized and washed with Hank��s balanced salt solution (HBSS) 3 times and counted using trypan blue (Sigma) exclusion dye. Single cell suspensions of 5 �� 104 cells (> 90% viability) in 100 ��l were injected into the mammary fat pad. Twice a week tumor size was measured using digital calipers (Fisher Scientific, Pittsburgh, PA). Tumor volume was calculated according to the formula: volume = W2 �� L/2, where W = short diameter and L = long diameter. PRDX5 Mice were injected intraperitoneally with MAZ51 at 10 MPK, 5 MPK or vehicle control (dimethyl sulfoxide) on days 1-7 and 14-21 following tumor inoculation. One week later mice were euthanized and primary tumor tissue, lymph nodes, lungs, and spleens were removed. Samples were processed by formalin fixation with subsequent embedding in paraffin, cryofrozen in OCT, frozen in liquid nitrogen or processed for isolation of cells. For experimental lung metastasis, Cl66 cells (5 �� 105 cells/0.1 ml HBSS/animal) were injected intravenously. The mice were euthanized when moribund. The lungs were removed, rinsed in water, and fixed in Bouin��s solution for 24 h to facilitate counting MCC950 concentration of tumor nodules. The number of surface tumor nodules was counted using a dissecting microscope. Sections of the lungs were stained with hematoxylin and eosin to confirm that the nodules were breast cancer. Survival data were collected from mice injected intravenously. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center. Immunohistochemical (IHC) analysis Protein expression was analyzed in paraffin-embedded primary tumors. Briefly, 4 ��m thick sections see more of paraffin blocks were processed for antigen retrieval by microwave citrate buffer treatment. The slides were processed for evaluation of apoptosis using the DeadEnd Colorimetric Apoptosis kit (Promega, Madison, WI) or for proliferation using an anti-PCNA (Santa Cruz Biotechnology) antibody. PCNA immunoreactivity was detected using the ABC Elite kit (Vector Laboratories) and DAB substrate (Vector Laboratories) kit per the manufacturer��s instructions. A reddish brown precipitate in the cytoplasm indicated a positive reaction. Negative controls used all reagents except the primary antibody. Intensity of staining was measured by two independent observers that examined each slide. Statistical analysis SPSS for Windows (SPSS Inc. Chicago, IL) was used for the independent samples t-test (two-tailed) to compare means. In vivo analysis was performed using Mann-Whitney U-test of significance. A value of p