At the end of the study, mice were killed and the tumors collected and weighed (B). reduces tumor growth. Similarly, the deletion of in mice protects against colon cancer in two different experimental models (inflammation-associated colon cancer and genetically driven colon cancer). In colon cancer cells, expression of the transporter is usually reduced by Wnt antagonist or by silencing of -catenin whereas Wnt agonist or overexpression of -catenin shows the opposite effect. Finally, SLC6A14 as a target for -catenin is usually confirmed by chromatin immunoprecipitation. These studies demonstrate that SLC6A14 plays a critical role in the promotion of colon cancer and that its up-regulation in cancer involves Wnt signaling. These findings identify SLC6A14 as a promising drug target for the treatment of colon cancer. mice were generated in our laboratory and have been used in a previously published study on the role of this transporter in breast cancer . This mouse line is usually on C57BL/6 background. mice on C57BL/6 background were obtained from Jackson Laboratory (Bar Harbor, ME, U.S.A.). The mice were maintained in a temperature-, humidity- and light-controlled environment in the animal facility at Texas Tech University Health Sciences Center (TTUHSC). The mice had access to water and rodent diet ad libitum. Age- and gender-matched control mice were used with the experimental groups. All experimental procedures were approved by the TTUHSC Institutional Animal Care and Use Committee (protocol Cefonicid sodium number, 17004). At the termination of the experiments, mice were killed by cervical dislocation under CO2 anesthesia in accordance with the guidelines from the American Veterinary Medical Association. Patient-derived xenografts The patient-derived xenografts (PDXs) were obtained from TXCCR (Texas Cancer Cell Repository) at TTUHSC Cancer Center (www.TXCCR.org). This center establishes the biorepository of PDXs and PDX-derived cell lines from primary clinical samples. All PDXs samples used in this study were from human colonic adenocarcinoma patients. The protocol had approval from the Institutional Review Board. Cell culture Normal human colonic epithelial cell line CCD841, human colon cancer cell lines (HCT116, HT29, Colo201, Colo205, SW480, SW620, KM12C, KM12L4, Caco2, and LS174T) and the mouse colon cancer cell line MC-38 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, U.S.A.). The cell lines were cultured in respective culture medium recommended by ATCC; culture media (Corning Life Sciences, Corning, NY, U.S.A.) were supplemented with 10% fetal bovine serum (Fisher Scientific, Pittsburgh, PA, U.S.A.) and 1% penicillin/streptomycin (Corning Life Sciences, Corning, NY, U.S.A.). HEK293FT cells were used for packaging lentivirus with plasmid and were maintained in DMEM, supplemented with 4.5?g/l glucose, l-glutamine, and sodium pyruvate, 10% FBS and 1% penicillin/streptomycin. Antibodies Anti-mTOR (#2983S), anti-P-mTOR (#5536S), anti-S6K (#9202S), anti-P-S6K (#9204S), anti-LC3A/B (#4108S) anti–catenin (#8814S), anti-Cyclin D1 (#2922S), anti-TCF4 (#2569S), and anti-IgG (#2729S) antibodies were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.). Anti-SLC6A14 (#A10582) polyclonal antibody was obtained from Abclonal. Anti–actin (C4, sc-47778) monoclonal antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, U.S.A.). Horseradish peroxidase-conjugated goat anti-rabbit IgG (#1706515) and goat anti-mouse IgG (#1706516) were purchased from Bio-Rad Laboratories (Hercules, CA, U.S.A.). Cefonicid sodium Analysis of gene expression datasets Three datasets with accession number “type”:”entrez-geo”,”attrs”:”text”:”GSE9348″,”term_id”:”9348″GSE9348 , “type”:”entrez-geo”,”attrs”:”text”:”GSE33113″,”term_id”:”33113″GSE33113 , and “type”:”entrez-geo”,”attrs”:”text”:”GSE34053″,”term_id”:”34053″GSE34053  were retrieved from publicly available gene expression omnibus database. The gene expression profiling of these datasets is based on the platform SLIT1 [HG-U133_Plus_2] Affymetrix Human Cefonicid sodium Genome U133 plus 2.0. Additionally, Illumina HiSeq_RNASeqV2 mRNA expression data for colon adenocarcinoma (COAD) were obtained from The Cancer Genome Atlas (TCGA) data portal. Samples were grouped as tumor and normal tissue and compared for gene expression. The student’s promoter (Supplementary Table S1). Xenograft of human colon cancer cells in immunosuppressed nude mice Male athymic BALB/c nude mice (8-weeks-old) were obtained from the Jackson laboratory and acclimatized with the environment before initiating the experiment. Mice were dived into two groups (control and treatment) with 5 mice in each group. The control group was provided with sucrose-water and treatment group with -MT (2?mg/ml) in sucrose-water 7 days prior to cancer cell injection. -MT was used as the d/l enantiomeric mixture. At day 0, both groups of mice were Cefonicid sodium subcutaneously injected with SLC6A14-positive human colon cancer cell line LS174?T (1??106 cells/mouse). Mice in the treatment Cefonicid sodium group continued to receive -MT in sucrose-water and the control group sucrose-water throughout the experiment..