A human Antibody Microarray 720 slides kit was pur chased from SPRING BIOSCIENCE. Briefly, the mem branes were blocked with a blocking Pazopanib Sigma buffer, and then 0. 1 mg Biotin Labeled Protein Sample was added and incubated at room temperature for 2 h. The membranes were washed, and 1 ml of Streptavidin Solution was added and incubated at room temperature for 45 min. The membranes were incubated with 1 ml of Detection Antibody Cy3 at room temperature for 45 min. The slides were e posed to film and processed by autoradiography. MicroRNA and mRNA detection QRT PCR assays were performed for measurement of the e pression levels of primary, precursor and mature miRNAs. Briefly, total RNA was e tracted with a mir Vana miRNA Isolation Kit and subjected to reverse transcription with the Reverse Transcription kit.

QRT PCR was performed with the Rotogene 3000 real time PCR system. For detection of mature miRNAs, Hairpin it miRNAs Real Time PCR Quantita tion Kit was used in accordance with the manufacturers protocol. Results were normal ized to U6 snRNA. For measurement of the primary and precursor miRNA e pression, real time PCR was performed using the SYBR method and b actin RNA was used for normalization. Bioinformatic prediction of miR 145 targets Putative miR 145 binding sites in DFF45 genomic sequence were predicted by the RNA22 program based on minimizing folding energy and ma imizing number of paired up bases in heteroduple . Plasmid construction Though bioinformatic analysis, the putative binding site of miR 145 was chemically synthesized and cloned into pGL3 control vector at ba1 site.

To con struct the DFF45 854 Wild vector, the entire region of DFF45 was amplified from the cDNA of LS17 and then cloned into the pGL3 control vector at ba1 site. To create the DFF45 854 Mutation vector, seven nucleotides were changed for the reporter construct. Luciferase assay LS174T cells or normal colon cells were plated in tripli cate wells of a 24 well plates and transfected with luci ferase reporters fused with putative binding site for miR 145, and miR 145 mimic inhibitor. Transfection efficiency was corrected by a renilla luciferase vector. The cells were harvested for luci ferase assays 24 hour after transfection. The Dual Luciferase Reporter Assay System was used to measure the reporter activity according the manufac turers protocol.

Western blotting assay Protein concentration was measured using Pierce BCA Protein Assay Reagent. Cell lysates were electrophoresed through 10% polyacrylamide gels and transferred to a NC membrane. The membrance was incubated with DFF45 antibody or p53 antibody. Secondary antibodies were labeled with IRDyes. Signals were visualized using an Odyssey Infrared Imaging System. Nuclear DNA fragmentation assay LS174T Batimastat cells and normal colon cells were treated with the indicated chemicals for appropriate time point. Cells were incubated in lysis buffer at 37 C for 30 min.