Cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin solu tion at 37 C in a humidified 5% CO2 atmo sphere. JP was obtained from Joyant Pharmaceuticals, Gem was purchased from Eli Lilly Cor porations, doxorubicin was pur chased from Ben Venue Laboratories and docetaxel was purchased from Sanofi aventis. Cell viability assay In vitro cell viability of PDAC cell lines was evaluated by using the colorimetric WST 1 reagent as described earlier. The assay is based on the ability of viable cells to cleave the sulfonated tetrazolium salt WST 1 2 2H 5 tetrazolio 1,3 benzene disulfo nate by mitochondrial dehydrogenases. Briefly, PDAC cells were plated in a 96 well plate in regular growth medium and after 16 hours the med ium was replaced with 2% FBS containing medium.

After 5 hours incubation the cells were treated with JP, Gem, Dox or DT, either alone or in combination. After additional incubation of 72 hours, 10 ul WST 1 reagent was added in each well followed by incubation for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Western blot analysis A monolayer of cells at 75 to 80% confluence was placed in 2% FBS containing medium for at least 5 hour before treatment with JP, gemcitabine for 24 hours. Cells were lysed and equal amounts of total protein were separated by SDS PAGE and trans ferred to PVDF membranes. The membranes were blocked for 1 hour at room tem perature with gentle shaking in TBS T, 150 mM NaCl, 0. 05% Tween 20.

The membranes were incubated overnight at 4 C with the following antibodies phospho JNK, poly poly merase 1 , a tubulin or GAPDH. The membranes were then incubated with corresponding HRP conju gated secondary antibodies for 1 hour at room temperature. Specific bands were detected using the enhanced chemilumines cence reagent on autoradiographic film and quantitated by densitometry. Apoptosis assay by Annexin V FITC and propidium iodide staining Effect of JP and Gem on AsPC 1 cell apoptosis was detected by using the Annexin V FITC apoptosis detec tion kit as per manufacturers protocol. Briefly, AsPC 1 cells were grown in 10% serum contain ing medium up to 80% confluence, medium was chan ged to 2% serum containing medium and incubated for 5 hours and then treated with JP and Gem for 12 hours.

After incubation, cells were trypsi nized and suspended in 1 ml of low serum medium. From each cell suspension 2 105 cell were centrifuged and re suspended in 500 ul 1X binding buffer. In each sample 5 ul of Annexin V FITC and 5 ul of PI was added, incubated for 5 minutes in dark and immediately analyzed by flow cytometry. The annexin V FITC positive and PI negative Dacomitinib cells were considered to be early apoptotic cells. Animal Studies All animal studies were performed in accordance with the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center.