The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE67 partner repository with the dataset identifier PXD016799. Proliferation index is an founded marker of tumor aggressiveness39,41. Its measurement allowed us to conclude that MS023 treatment was able to significantly reduce the proliferative NOX1 potential of HT-29 xenograft, suggesting differentiation changes in the treated tumors. Our data demonstrates MS023 was able to reduce tumor growth and proliferation in vivo through differentiation of malignant cellsdemonstrating a tractable model of pharmacological manipulation of colon cancer differentiation, with medical implications. Methods Cells tradition and handling HT-29, HCT-116, HCT-15, SW-620, Colo-205 colon cancer and CCD-841 normal colon epithelial cell lines were purchased from ATCC. GFP expressing HT-29 cells were kindly provided by Dr. Z. Elazar lab, the Weizmann Institute of Technology, Rehovot, Israel. Malignancy cells were cultivated in Roswell Park Memorial Institute growth medium (RPMI-1640, Biological Industries Israel, # 01-101-1A) and normal cells in Eagle’s Minimum amount Essential Medium (EMEM, Sigma-Aldrich, #”type”:”entrez-nucleotide”,”attrs”:”text”:”M22790″,”term_id”:”187686″,”term_text”:”M22790″M22790) mediums supplemented with 10% warmth inactivated Fetal Bovine Serum (FBS), EGFR-IN-7 1?mM l-glutamine and EGFR-IN-7 1% penicillin/streptomycin solution (all from Biological Industries). Exclusion of Mycoplasma contamination was monitored and carried out by test with Mycoalert kit (LONZA, #BELT07-218). HTS products Screen and hit validation experiments were performed using the HTS products: Echo555 Acoustic transfer system (Labcyte, Germany), Combi MultiDrop (Thermo Fischer Scientific), Washer Dispenser II (GNF, San Diego, CA, USA), EL406 Microplate Washer Dispenser (BioTek, Winooski, VT, USA), Bravo Automated Liquid Handling (Agilent, Santa Clara, CA, USA). Luminescence signals were measured by luminescence module of PheraStar FS plate reader (BMG Labtech, Ortenberg, Germany). Acumen laser scanner (TTP Labtech, Hertfordshire, United Kingdom) and ImagExpress EGFR-IN-7 Micro XL high content material microscope (Molecular Products, Sunnyvale, CA, USA) were used to acquire and analyze images. Testing process and CDP/CTG multiplex luminescence assay 5760 compounds from Selleck Chemicals Bioactive, MEGxp Pure natural compounds (Analyticon), Drug-Like Arranged (DLS, Enamine) and whole SGC Epigenetic Chemical Probe Collection chemical libraries (30 compounds) were screened using HT-29 cells by previously explained multiplex method for detection and normalization of ALP level in cells29. Briefly, 250 cells/well were plated in 50?l of growth medium into white colored/white colored 384-well TC plate (Greiner, #781080) and treated with either compounds or settings (sodium butyrate, DMSO). After 5?days cells were washed 4 instances, lysed, and two consequent luminescent signals (CDP for alkaline phosphatase activity and CTG for cell viability) were measured. Related procedure was used in all follow up experiments, where ALP activity and cell viability were measured. Data analysis, normalization and statistics Screening and hit validation data were plotted and analyzed using GeneData 12 and 15 (Basel, Switzerland), and Collaborative Drug Discover (CDD) softwares (Cambridge, United Kingdom). Additional statistical significances were evaluated by two tailed t test, *adjusted value?