Eight patients were treated with Intensity modulated radiation therapy at 50 Gy and responses were evaluated via computed tom ography. Five patients who have stable disease or pro gressive disease were resistant to IMRT among total 8 patients. The biopsies were taken by tru cut needle from these five radiotherapy resistant patients. None of the sub jects received other biotherapy selleck products or chemotherapy treat ments. The study was approved by the ethics committees of the First Hospital of Jilin University and the Fourth Military Medical University. Written informed consents were also obtained from all subjects before study. Cell culture and sulforhodamine B assay Human pancreatic cancer cells PANC 1, Capan 2 and BxPC 3 purchased from National Rodent Laboratory Ani mal Resource were grown as previously described.

Briefly, these cell lines were cultured and maintained in exponential growth in Dulbeccos modified Eagles medium containing 100 IU ml penicillin, 100 ug ml streptomycin, 20 mM glutamine and 10% heat inactivated FCS in a humidified atmosphere Inhibitors,Modulators,Libraries of 5% CO2 at 37 C. For sul forhodamine B assay, the exponential Inhibitors,Modulators,Libraries growing cells were seeded at 6 8 103 well in 96 well plates and cultured overnight. Cells were treated with radiation alone or combined with AZD8055. AZD8055 was added to cultured cells and radiation was applied 4 h later in single doses of 1, 2. 5, 5 or 10 Gy. The cells were irradiated using an X ray machine at 320 kV, 10 mA with a 2 mm aluminum filter, and the dose rate was 2 Gy min. Cells were then cultured at 37 C for 48 h and the surviving fractions were determined using SRB assay as previously described.

The absorbance was measured with a spectrophotometer at 510 nm and cell growth inhibition was calculated by using the equation cell viability 100%, in which At and Ac represent the absorbance in treated and control cultures respectively, as described previously. Cell lysate and Western blot assay Cells were lysed in Inhibitors,Modulators,Libraries ice cold EBC buffer, 20 uM sodium orthovanadate, 1 Protease Inhibitors, 1 Phosphatase Inhibitors and proteins were quantified and subjected to SDS PAGE electrophoresis, followed by protein trans fer to nitrocellulose membranes. Inhibitors,Modulators,Libraries The membranes were incubated with the primary and secondary antibodies, then developed by chemiluminescence.

RNA isolation and quantitative real time PCR Total RNA was isolated from cells using Trizol, 1 10 ug of RNA was used to synthesize cDNA with Super Script II First Strand Synthesis System or TaqMan MicroRNA Reverse Transcription Kit. Aliquots of the reaction Inhibitors,Modulators,Libraries mixture were used for real time PCR with Power SYBR Green PCR Master Mix or with the TaqMan any other enquiries 2 Universal PCR Master Mix. The reaction conditions 50 C for 20 s, 95 C for 10 min followed by 40 cycles of 95 C for 15 s, 60 C for 1 min. All real time PCR experiments were performed in triplicate. A melting curve was obtained to verify the presence of a single amplicon.