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Serum hormones After study termination, serum insulin and leptin levels were analyzed using Lincoplex? bead-based multiplexed assays (Millipore, Billerica, MA, USA; MADPK-71 K-07). Serum adiponectin and IGF-1 were quantified by [http://www.selleckchem.com/products/ipi-145-ink1197.html learn more] singleplex assay kits (Millipore; MADPK-71 K-ADPN and RMIGF187K, respectively). All assays were analyzed using a BioRad Bioplex? analyzer (BioRad, Hercules, CA, USA) according to manufacturer��s directions. Immunohistochemical analyses Formalin-fixed tissues were embedded in paraffin, cut into 4-��m thick sections, and processed for immunohistochemistry at the Histology Core Laboratory at The U.T. M.D. Anderson Cancer Center, Science Park Research Division (Smithville, TX, USA). Antibodies used for immunohistochemistry were optimized by core personnel [http://en.wikipedia.org/wiki/PRDX4 PRDX4] using positive and negative controls for each analysis. Slides were deparaffinized in xylene and sequentially rehydrated in ethanol to water. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 minutes. Antigen retrieval required microwaving slides with 10 mM citrate buffer. Nonspecific binding was blocked by treating sections with Biocare blocking reagent (Biocare Medical, Concord, CA, USA) for 30 minutes at room temperature, followed by incubation with primary antibody diluted in blocking buffer overnight at 4��C. The following primary antibodies and dilutions were used: phospho-S6 ribosomal proteinS235/236 (Cell Signaling, Danvers, MA, USA; 1:100); phospho-mTORSer2448 (Cell Signaling; 1:50); phospho-ACCSer79 (Cell Signaling; 1:50); Ki-67 (Dako, Carpinteria, CA, USA; 1:200); cyclin D1 (Santa [http://www.selleckchem.com/products/Bleomycin-sulfate.html Bleomycin cost] Cruz Biotechnology, Santa Cruz, CA, USA; 1:500); and cleaved caspase-3 (R&D Systems, Minneapolis, MN, USA; 1:500). Slides were washed twice in PBS, incubated for 30 minutes with secondary antibody, washed two times with PBS, stained with diaminobenzidine (BioGenex, Fremont, CA, USA) and counterstained with hematoxylin. Images were captured by the Aperio ScanScope XT (Aperio Technologies, Vista, CA, USA) and staining was quantified using the Aperio ImageScope (Aperio Technologies). For Ki-67, phospho-mTOR, and cyclin D1 quantification, automated algorithms were used to determine negative or positive nuclear staining. The percentage of positive cells was obtained with 20�� objective in pancreatic tumor sections. Positive staining was defined as 1+, 2+, and 3+ for cyclin D1 and phospho-ACC. Positive staining for phospho-mTOR was defined as only 2+ and 3+ due to its high baseline phosphorylation. Cleaved caspase-3 (CC3) was quantified as the average area of positively stained cells, with positive staining defined as at least 70% of a 100 ��m?��?100 ��m section. For all proteins, the positive staining was averaged per group (n?=?5/group).