Supplies and Reagents ISC 4 was synthesized following a process recently manufactured by Sharma et al.. Other reagents obtained from: 5 FU Acros Organics, API 2, and PBITC. Cell culture reagents: HT29, SW480, HCT116, and Fugene 6 reagent and SW620 cells Hedgehog inhibitor Vismodegib. Antibodies were ordered from Santa Cruz Biotechnology, Santa Cruz, CA, Amersham, Piscataway, NJ, and Cell-signaling Systems, Boston, MA. Cell culture Human a cancerous colon cells were cultured in RPMI containing 10% FBS and Pen/Strep at 5% CO2 and 37 C. HT29 cells were transfected with either rat par 4 cDNA in pCB6 , with the individual Par 4 clone in pCMVA6 AC, or with empty vector using Fugene 6. Individual Par 4 was obtained from Origene. Transfectants were chosen with G418 and cities assayed and expanded for Par 4 term. Immunoprecipitation and Western blotting Antibodies applied were: Par 4 rabbit polyclonal, Caspase 9 rabbit polyclonal, Caspase 8 mouse monoclonal, and B actin mouse monoclonal. Cells were grown to 800-916 neuroendocrine system confluence. Plates were washed with PBS and the cells were lysed into lysis buffer. In case of mouse tissues, snapfrozen tissues were homogenized in lysis buffer employing a Fisher Scientific PowerGen homogenizer. The proteins were filled similarly onto 10 percent polyacrylamide fits in and quantified according to the Bradford Assay. For immunoprecipitation, 100 ug protein were incubated with 50 ul Dynabeads conjugated to 14 3 goat polyclonal antibody. Beads were washed and proteins eluted. Proteins were electrophoresed at 150 v and transferred to nitrocellulose filters using a semi-dry blotter. Membranes were blocked with five minutes non-fat dry milk for just two h and incubated with primary antibody overnight. The blots were cleaned 3X in TBS Tween and incubated for 1 h in correct HRP conjugated secondary antibodies. Blots were washed and developed utilizing the ECL chemiluminescent set. HT29 cells, transfected with either Par 4 or empty vector, were handled with ISC 4. In Imatinib price vitro cytotoxic efficacy was measured using 3 2,5 diphenyltetrazolium bromide, a tetrazole cell viability assay. Naked mouse tests All mice were treated in line with the directions set forth by the Association for Assessment and Accreditation of Laboratory Animal Care. Forty 6 week old female athymic nude mice were injected in the proper flank with 107 wild-type HT29 cells and 20 of the mice were also injected in the left flank with 107 HT29 cells transfected to overexpress Par 4. Starting at seven days post injection, tumors were measured weekly with calipers. Tumor volume was determined using the equation, Half the mice were treated 3X weekly with ISC 4, at 3 PPM in 50 ul DMSO by intraperitoneal injection. Half of the ISC 4 treated mice were additionally treated by IP injection with 5 FU on day 28 after injection of cells.