All these findings strongly supported that IGFBP7 played a potential tumor suppressor role against colorectal carcinogenesis. In consistent with our findings, the tumor suppressor roles of IGFBP7 in cervical cancer[10], osteosarcoma[10, 11], prostate cancer[12, 13], and breast cancer[14] were discovered by other laboratories. The important function of IGFBP7 protein in CRC has elicited the need to further investigate the underlying mechanism. Proteomics represents a powerful approach to analyze alterations in protein expression in complex biological system. This approach has been used successfully in our lab to identify differentially expressed proteins between tissue of colorectal carcinoma, colon adenoma, and the normal

mucosa, which have potential Selleckchem VX-680 clinical interest [15, 16]. In this study, our main goal

was to identify proteins associated with IGFBP7 expression using the proteomics-based approach and further clarify the protein’s biological role. These findings will contribute to our understanding for the molecular mechanism responsible for IGFBP7′s tumor suppressive function in CRC. Methods Reagents Dulbecco’s Modified Eagle’s Medium(DMEM)was purchased from GIBCO Laboratories (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from HyClone Laboratories (Logan, UT, USA). Polyfect transfection reagent was purchased from QIAGEN (Hilden, Germany). G418 was purchased from Merck (Darmstadt, Germany). Immobiline Dry-Strips (17 cm, pH 3-10 NL), immobilized pH gradient (IPG) buffer, Dry-Strip cover fluid, urea, thiourea, ammonium bicarbonate and TSA HDAC sodium dodecyl sulfate/polyacrylamide ADP ribosylation factor gel electrophoresis (SDS-PAGE) standards were purchased from BioRad (Hercules, CA, USA). Dithiothreitol (DTT), trifluoroacetic acid (TFA), acrylamide, cellulose acetate nitrate (ACN), glycerol, glycine, iodoacetamide3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonic acid (CHAPS), bis-hydroxymethyl-oxazoline (Bis), tetramethylethylenediamine (TEMED), sodium dodecyl sulfate (SDS), tris-hydroxymethyl-aminomethane (Tris base), dimethylsulfoxide (DMSO), bovine serum albumin (BSA) and Coomassie brilliant blue (CBB R-250) were obtained from Sigma

Chemical (St. Louis, MO, USA). Cell lysis buffer, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, 60 kDa heat shock protein (HSP60) antibody, and horseradish peroxidase-linked second antibody were purchased from cell signaling Technology (Danvers, MA, USA). Recombinant human HSP60 protein and HSP60 ELISA kit were purchased from StressGen Biotechnologies (Victoria, Apoptosis inhibitor British Columbia, Canada). Cell culture and protein extraction Human colorectal carcinoma RKO cell lines were derived from the American Type Culture Collection (ATCC), maintained in DMEM supplemented with 10% FBS in a 37°C/5% CO2 atmosphere. RKO cells were transfected with either PcDNA3.1(IGFBP7) or an empty plasmid vector PcDNA3.1. Stable PcDNA3.1(IGFBP7)-RKO transfectants and PcDNA3.