Sequence range 11673–12146 2950–3101 881–1437 1715433–1715638 Product size (bp) 473 171 556 206 Table 3. List of primers used for genotyping. Primer name NCBI GeneBank accession IDs AdipoWT Neo NT_039624 User_PGKneobpA Sequence sequence 4575 bp DNA circular SYN 08/24/2007 MMTV/PyVT J02288 Tcrd NG_007044 doi:10.1371/journal.pone.0004968.t003 0.2 M Glycine-HCl, pH 2.5, which was neutralized with 10 ml of neutralization buffer (1 M Tris-HCl, pH 9.0). The elutants were subjected to 15% SDS-PAGE and Western blotting analysis, or enzyme activity measurement as described below. For Western Blotting, fifty micrograms of proteins derived from cell or tissue lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Following blocking, membranes were probed with various primary antibodies to determine different levels of protein expressions. Immunoreactive antibody-antigen complexes were visualized with the enhanced chemiluminescence reagents from GE Healthcare (Uppsala, Sweden). [3H]-thymidine incorporation assay for cell proliferation measurement 56104 of isolated primary tumor cells were seeded into each of the 24-well culture plate and allowed a period of at least 24 hours for cell settlement and attachment. After being treated under 3 different conditions, 1 mCi/ml of [ H-methyl] thymidine was added into each well for 6 hours of incorporation. At the end of experiment, the culture media were removed and cells washed twice with cold PBS. DNA was precipitated with 0.5% trichloroacetic acid for 30 min. Air-dried precipitates were then solubilized with 0.2 mol/l NaOH, neutralized with 0.2 mol/l 3 HCl, and incorporated [ H]-thymidine was quantified with a liquid scintillation counter (Backman LS6500). Quantitative RT-PCR Total RNA was isolated from primary tumor cells and used for the synthesis of cDNA. Quantitative RT-PCR was performed using SYBRH GreenERTM qPCR Supermix (Invitrogen, Carls- bad, CA). The reactions were carried out on a 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Quan- tification was achieved using Ct values that were normalized with 18S RNA as internal control. The primers were listed in Table 4. Beta-catenin/T-cell factor-lymphoid enhancer factor-1 (TCF-LEF) and thioredoxin reductase 1 (TrxR1) transcription reporter assay Nuclear activities of endogenous beta-catenin were analyzed by the TOPflash/FOPflash reporter system as described previously [28]. To normalize transfection efficiency in the reporter assays, cells were cotransfected with pRL-TK plasmid, which contains a functional Renilla luciferase gene cloned downstream of a herpes simplex virus thymidine kinase promoter (Promega, Madison, WI). Adiponectin and Breast Cancer Primer sequences (F) 59- CCA GAG AAC AAC GAA CAA GGA- 39 (R) 59 – CGA ATG GGT ACA TTG GGA AC- 39 (F) 59 – TGA ATG AAC TGC AGG ACG AG- 39 (R) 59 – ATA CTT TCT CGG CAG GAG CA- 39 (F) 59- GGA AGC AAG TAC TTC ACA AGG G- 39 (R) 59- GGA AAG TCA CTA GGA GCA GGG- 39 (F) 59- CAA ATG TTG CTT GTC TGG TG- 39 (R)59 GTC AGT CGA GTG CAC AGT TT- 39 The luciferase reporter containing a human thioredoxin reductase promoter region (from 2386 bp to +218 bp, pGL3-TR) was constructed into a firefly-luciferase pGL3-basic Vector (Promega, Madison, WI). The assay was carried out as described above with the original unmodified pGL3-basic vector as a negative control. Luminescence was measured using a Bright-GloTM Luciferase Assay System (Promega, Madison, WI) on Lumat LB9507, and normalized to control Renilla luciferase signal. Luciferase activity was calculated against the negative control signals and fold differences were compared among groups in separate assays. Measurement of PTEN lipid phosphatase activities The lipid phosphatase activity of PTEN was measured as described previously with slight modifications [40]. Phosphatase reactions were performed in 25 ml assay buffer (100 mM Tris-HCl pH 8, 10 mM DTT, and 200 mM water-soluble diC8-PIP3) with 25 ml sample. PTEN proteins immunoprecipitated on protein G- Sepharose beads (Pierce Biotechnology, Rockford, IL) were washed twice in a low stringency buffer (20 mM HEPES, pH 7.7, 50 mM NaCl, 0.1 mM EDTA and 2.5 mM MgCl2) and once in the phosphatase assay buffer lacking PIP3. Reactions were done in a 96- well plate with an incubation period of 40 minutes at 37uC. The release of phosphate from the substrate was measured in a colorimetric assay by using the Biomol Green Reagent in accordance with the instructions of the manufacturer. The absorbance at 620 nm was measured with a mQuant MQX200 microplate reader (Biotek Instruments, Inc., Highland Park, VT). A standard curve was performed in each assay, and the amount of free phosphate was calculated from the standard curve line-fit data. Measurement of TrxR1 and Trx1 activities The assays for measuring the activities of TrxR and Trx were performed in 96-well plates using an insulin reduction endpoint assay as described previously [41] with slight modification. For determi- nation of TrxR1 activity, 25 mg of the primary tumor lysates were mixed thoroughly with a 50 ml reaction buffer containing 55 mM HEPES, pH 7.6, 0.02 mM insulin, 0.4 mM NADPH, 2 mM EDTA and 2 mM human Trx1 in the mQuant MQX200 microplate reader (Biotek Instruments, Inc). Reaction solutions without human Trx1 were used as the control. After performing a 20-min incubation at 37uC, 200 ml of 1 mM DTNB in 6 M guanidine hydrochloride solution was added to stop the reaction. The free thiols of the reduced insulin were determined by DTNB reduction, and the activity of TrxR was represented as the absorbance at wavelength 412 nm, where 1 mole of NADPH reduced 1 mole of disulfide, giving rise to 2 PLoS ONE | www.plosone.org 13 March 2009 | Volume 4 | Issue 3 | e4968