This study evaluated masitinib using in vitro and in vivo models of human pancreatic cancer, both as an individual agent and in combination with gemcitabine, with the objective of establishing proof of concept. Molecular elements were Raf inhibition investigated via gene expression profiling. Masitinib was prepared from powder as a 10 or 20 mM stock solution in dimethyl sulfoxide and located at 280uC. Gemcitabine was obtained as a dust and dissolved in sterile 0. 9% NaCl solution and saved as aliquots at 280uC. Clean dilutions were prepared for every single experiment. Pancreatic cancer cell lines were obtained from Dr. Juan Iovanna. Cells were preserved in RPMI or DMEM medium containing Glutamax 1, supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% foetal calf serum. Expression of tyrosine kinases was determined by RT PCR using Hot Star Taq in a Thermal purchase GDC-0068 Cycler. All RT PCR primer sequences used in this study are shown in the Information. Mia Paca 2 cells were treated for 6 hours with increasing levels of masitinib in DMEM medium with 0. 5% serum. Cells were then positioned on ice, washed in PBS, and lysed in 200 ml of ice cold HNTG barrier in the presence of 100 mM Na3VO4 and protease inhibitors. Proteins were resolved by SDS PAGE 10%, adopted by western blotting and immunostaining. The next key antibodies were used: rabbit anti phospho GRB2 antibody, and anti phosphotyrosine antibody. Major antibodies were detected with 1:10,000 horseradish peroxidase conjugated anti rabbit antibody or 1:20,000 horseradish peroxidase conjugated anti mouse antibody. Immunoreactive bands were found using superior chemiluminescent Cellular differentiation reagents. Cytotoxicity of masitinib and gemcitabine was assessed utilizing a WST 1 proliferation/survival analysis in growth medium containing 1% FCS. Treatment was started with the addition of the drug. For mix treatment, cells were first resuspended in medium containing 0, 5 or 10 mM masitinib and incubated overnight before gemcitabine improvement. After 72 hours, WST 1 reagent was added and incubated with the cells for 4 hours before absorbance measurement at 450 nm within an EL800 Universal Microplate Reader. Media alone was used as an empty and growth in the absence of drug served as a control. Email address details are representative of three to four tests. The masitinib sensitisation list is the ratio of the IC50 of gemcitabine against the IC50 of the drug combination. Male Nog SCID mice were obtained from an internal breeding system and were housed at your pet care model SCEA of the Dalcetrapib molecular weight Centre de Recherche en Cance?rologie de Marseille U891 under specific pathogen free conditions at 2061uC in a 12 hour light/12 hour dark period and ad libitum access to food and filtered water. This study was accepted by the ethical review board at the Centre de Recherche en Cancerolgie de Marseille and completed in compliance with the INSERM ethical guidelines of animal experimentation.