Appropriate positive and negative controls were established. The sections were counterstained with hematoxylin in the end. The positive expression of vimentin was yellow stain in the cytoplasm of melanoma cells. Ten “”hot spots”" under high-power fields were selected
randomly, and 100 cells per field were counted. The average percentage of positively stained cells of 10 fields was converted into a score as follows: 0 for < 20%, 1 for < 40%, 2 for < 60%, and JNJ-26481585 datasheet 3 for > 60%. A score between 2 and 3 was considered to be strong expression. Statistic analysis The statistical analysis was conducted using SPSS version 13.0 (SPSS, Chicago, IL, USA). P-value less than 0.05 was defined as significant level. Values are shown as mean ± SD or percentages. The χ2 test, the Student’s t-test and the Mann-Whitney test were used in our study. Kaplan-Meier survival analysis and log-rank test were performed to compare the survival time between each group. Multivariate survival analysis was performed using the Cox proportional hazards model. Results 2D-DIGE Images of Proteins Protein profiles which
were potentially involved in metastasis were analyzed by 2D-DIGE which was repeated independently for three times under identical condition. The threshold of proteins differential expression was set at great than 2-fold, and the P value of < 0.01 of t-test was regarded as statistical MRT67307 manufacturer significance. Thirty spots across all images were differential significantly. They were subsequently excised, subjected ADP ribosylation factor to trypsin digestion in-gel and analyzed by MALDI-TOF/TOF- MS. Thirteen proteins of them were successfully identified by PMF analysis and peptide sequences analysis in the NCBInr database (Table 2). Of the 13 protein spots, 11 were higher abundance and 2 were at lower levels in the metastatic group. Highly expressed proteins in B16M group included cytoskeleton/structure proteins (vimentin, gamma-actin, β-actin, laminin binding protein), the chaperone family of proteins (heavy-chain binding protein, Bip),
immunoproteasome assembly (proteasome activator REG alpha) and others involved in glycolysis activity (PGK1, enolase, TPI, human AZD0156 supplier skeletal muscle GAPDH) and protein transport (myoglobin). MALDI-TOF/TOF-MS analysis and database matching identified spot 625 was vimentin with high sequence coverage and mass accuracy (Figure 1A-C). Table 2 The list of differential proteins identified by MS spot no. Acession number identified protein average ratio M.W(Da) PI Protein coverage Protein score C.I.% Functional classification 520 gi|17389985 MB protein [Homo sapiens] myoglobin 2.13 10863 9.24 40% 100 Transport 597 gi|31873302 hypothetical protein [Homo sapiens] -3.24 47063 7.57 8% 99.993 625 gi|47115317 VIM [Homo sapiens] 2.06 53547 5.09 27% 97.337 Cytoskeleton 641 gi|16552261 unnamed protein product [Homo sapiens] -2.7 47459 5.01 42% 100 687 gi|178045 gamma-actin[Homo sapiens] 2.26 25862 5.
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