Murine pancreas cancer cell lines K399, K389, K375, K162, K152, and K518 were developed ex vivo from tumors of mice overexpressing K rasG12D and TGF b knockout, and were obtained from Dr. H. Moses. The formulation and the in vivo dosing Belnacasan (VX-765) schedule of g secretase inhibitor MRK003 were provided by Merck Co.Inc, and were described previously. The mTOR inhibitor rapamycin and the Rock1 inhibitor Y27632 were obtained from Sigma Aldrich and CalBiochem, respec tively. The g secretase inhibitor DAPT L alanyl] S phenylglycine t butyl ester was also obtained from Sigma Aldrich. The dominant negative Notch3 and VC constructs were trans fected into BxPC3 and selected with G418, as previously described. Notch3 siRNA3 sequences were also described previously.

TMA Construction, TMA Slide Preparation De identified tumor and adjacent normal tissues were obtained under an IRB approved Inhibitors,Modulators,Libraries protocol at Vanderbilt University Medical Center. Before constructing a TMA block, Inhibitors,Modulators,Libraries serial 5 um sections were cut from each donor block. One of these sections was stained with H E for marking morphologically representative areas of the tumor. Using a Beecher Instruments Tissue Arrayer, tissue cylinders with a diameter of 0. 6 mm were punched from the four targeted areas in each donor block and deposited into a 9 14 TMA block, which contained 76 cores of adenoma tissue and 50 cores of adjacent, non malignant tissue as controls. The TMA blocks were warmed to 36 C for 30 minutes, and multiple serial 5 um sections were cut and placed on charged slides. Antibodies The Notch3 antibody 1E4 was used for immunohistochemistry, and the method was described previously.

Inhibitors,Modulators,Libraries Jagged1 and Notch4 were purchased from Santa Cruz, whereas Notch1, DLL4 and Notch2 antibodies were obtained from Cell Sig naling Technology, Sigma Aldrich, and the Developmen tal Studies Hybridoma Bank, respectively. Human EGFR antibody was purchased from Zymed. The IHC staining was scored on a composite scale of 0 to 3 by two independent observers, Inhibitors,Modulators,Libraries including one pathologist. In case of disagreement, the decision was deferred to the pathologist. The tumors that scored 2 or better were considered positive. For immunoblotting, Inhibitors,Modulators,Libraries Notch1, Notch3, phospho Akt, total Akt, PTEN, pPTEN, RhoA, Rock1, cdc42, Bcl xL, Bcl 2 and PARP were obtained from Cell Signaling Technology.

For specific use in murine cell lines, Jagged1, Notch1, and Notch3 were obtained from Santa Cruz, and Notch2 and Notch4 were purchased from DSHB and Orbigen, respectively. Real time RT PCR Total RNA was Brefeldin A solubility isolated from K399 cells using Sure Prep RNA Purification Kit. cDNA synthesis was carried out using iScript cDNA Synthesis Kit, accord ing to manufacturers recommendation. Proliferation Assays, Soft Agar and Cell Death Analysis Cells were plated into 96 well microtitre plates at 10% FCS and at 50% confluency in 200 ul DMEM.