Ls174T cells with inducible ��-catenin and KRAS shRNA were described previously [19]. The following sellekchem antibodies were used in this study: anti-KRAS (Abnova, clone 4F3, diluted 11000), anti-pygopus (Santa Cruz biotechnology, H-216, 1200), anti-myc (Santa Cruz biotechnology, 9E10, 1200), anti-actin (Sigma-Aldrich, 12000), anti-survivin (Santa Cruz biotechnology, D-8, 1200), anti-��-catenin (Millipore, 2H4A7, 11000). PKF115-584 was kindly provided by Novartis, Inc. (Basel, Switzerland). S-trans, trans-farnesylthiosalicylic acid (FTS) was synthesized as described [26]. Pyrvinium pamoate was purchased from Sigma-Aldrich. All inhibitors were dissolved in DMSO and stored in small aliquots at ?20��C.

Cell growth and cell death assays Cell growth and viability was assessed at the indicated times using the CellTiter 96? AQueous One Solution Cell Proliferation Assay System (Promega Corporation) following instructions, as previously reported [37]. To evaluate induction of apoptosis, the cells were seeded in 6-well plates overnight and then treated with inhibitors or vehicle. After 72 hours, the cells were detached by trypsin, washed, and apoptosis was determined using the Annexin V-FITC Apoptosis Detection Kit (Bender MedSystems), according to manufacturer’s instructions. All graphs and IC50 values were generated using the GraphPad software. Dual luciferase assay The cells in 6-well plate were treated with inhibitors or vehicle and transfected with 2 ��g of TOPflash or FOPflash plasmids and 0.1 ��g of phRL-CMV (encoding for Renilla luciferase, used as an internal control for transfection efficiency) using 3 ��l of FuGENETM 6 reagent.

After 24 hours, the cells were harvested, washed, and lysed. Luciferase signals were detected using the Dual-Luciferase? Reporter Assay System (Promega). Firefly luciferase intensity was normalized over Renilla luciferase signal. Active KRAS pull-down assay The cells were treated Carfilzomib with FTS or DMSO for 15 hours and then lysed in Magnesium Lysis Buffer (MLB: 25 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1% NP-40, 0.25% sodium deoxycholate, 10% glycerol) containing protease inhibitors (10 ��mol/L benzamidine-HCl and 10 ��g/mL each of aprotinin, leupeptin and pepstatin A). Lysates were clarified by centrifugation at maximum speed and quantified by Bradford assay. Equal amount of total proteins (1 mg) were then incubated with 10 ��g of Raf-1 RBD agarose beads (Millipore) for 45 minutes at 4��C on a rotating wheel. After 3 washes with MLB, the beads were resuspended in 2X Laemmli sample buffer, boiled and loaded on SDS-PAGE. Active, pulled-down KRAS was revealed by anti-KRAS antibody. Total KRAS (input) was evaluated from the crude lysate.