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Omission of primary antibody was used as a negative control. Frozen metastatic and nonmetastatic bones were crushed and processed for RNA extraction using the RNeasy Fibrous Tissue Mini Kit (Qiagen), according to the supplier's instructions. Complementary DNA was then obtained using MLV-1 reverse transcriptase (Qiagen). Serum tartrate-resistant acid phosphatase isoenzyme 5b (s-TRACP5b), an osteoclast specific enzyme, was measured by ELISA using a capture antibody raised against recombinant mouse TRACP5b (SBA Sciences, IDS, Paris, France).32 The intra-assay and interassay http://www.selleck.cn/products/carfilzomib-pr-171.html CVs were lower than 6.5 and 8%, respectively. Serum C-terminal cross-linked telopeptide of type I collagen (s-CTX-I), a biochemical marker of bone matrix degradation, was measured by a competitive ELISA using an antibody raised against an 8 amino acid sequence of type I collagen C-telopeptide (Nordic Bioscience, IDS). Intra-assay and interassay CVs were less than 9.5%. Serum type I collagen N-propeptide (s-PINP), a biochemical marker of bone formation, was measured by a competitive ELISA using an antibody raised against an 8 amino acid sequence of PINP (IDS).33 The intra-assay and interassay CVs were lower than 7.5 and 9.5%, respectively. Serum osteocalcin (s-OC), a marker of osteoblastic activity, was measured by a two-site immunoradiometric assay using antibodies raised against the mid region C-terminal portion and the N-terminal fragment of osteocalcin (Immunotopics, IDS). The intra-assay and interassay CV were less than 5 and 5.5%, respectively. All data were analyzed with the use of StatView software (version http://www.selleckchem.com/products/Metformin-hydrochloride(Glucophage).html 5.0; SAS Institute Inc., Cary, NC). Comparisons were carried out by performing a nonparametric Mann-Whitney U test. Correlations were evaluated by performing a nonparametric Spearman test. p values http://www.selleckchem.com/products/BIBF1120.html cancer cell lines (Fig. 1a). The presence of periostin protein in the cell lysate or in the supernatant of cultured human breast and prostate cancer cell lines was assessed by ELISA. Periostin was not detected in any of the cell lines, except for MC3T3-E1 cells, which were used here as a positive control (Fig. 1b). Western blot analysis of these samples with the anti-periostin antibody confirmed ELISA data; only MC3T3-E1 expressed periostin [Fig. 1b (inset); cancer cell lines were all negative and not shown].