Materials and Methods Materials Antibodies against PI3K p110-alpha (#4255), PI3K p85 (#4292), phospho-Akt (Ser473) (#9271), Akt (#9272), GSK3beta (#9315), phospho-GSK3beta (Ser9) (#9336) and phospho-beta- catenin (Ser33/37/Thr41) (#9561) were obtained from Cell Signaling Biotechnology (Beverly, MA). Anti-Trx1 (sc-20146), anti-TrxR1 (sc-28321), and anti-beta actin (sc-1615) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti- PTEN (MAB4037) was from Chemicon International, Inc. (Temecula, CA), anti-Cyclin D1 (CC12) was from CalBiochem- Novachem Crop. (San Diego, CA), and sheep anti-beta-catenin was from Symansis (Auckland, New Zealand). Pharmacological inhibitors, including Akt-1/2 inhibitor, PI3K p110alpha inhibitor PIK-75, PI3K p110beta inhibitor TGX221, and PI3K p110delta inhibitor IC87114 were provided by Dr Peter R. Shepherd [34,35]. The general PI3K inhibitor, LY294002, was from Cell Signaling Biotechnology. ImProm-IITM Reverse Transcription System and Bright-GloTM luciferase assay system were from Promega (Madison, WI). TOP/FOPflash (T-cell factor-lymphoid enhancer factor-1 (TCF-LEF) reporter plasmid) was from Upstate (Lake Plasid, NY). pGL-TrxR1 reporter plasmid was generated by cloning the proximal promoter of the human TrxR1 gene using the GenomeWalker kit from Clontech (Palo Alto, CA). The human TrxR1 and Trx1 were purchased from Sigma. The rat TrxR1 was purified from rat liver according to published procedure [36] and the purity confirmed by mass spectrometry analysis. Unless specified, all chemicals were obtained from Sigma- Aldrich Co. (St Louis, MO). Recombinant full length adiponectin (ADN) was produced as we described previously [28]. Establishment of the MMTV-PyVT transgenic mice haplodeficient in adiponectin expression FVB/N-Tg(MMTV-PyVT)634Mul/J transgenic mice were obtained from the Jackson Laboratory (Bar Harbor, Maine) [37]. Since the female PyVT transgenic mice were defective in litter delivery and lactation, all breedings were carried out using male PyVT transgenic mice. The male heterozygote PyVT(+/2) mice were cross-bred with female adiponectin knockout mice [38] and back-crossed for at least 12 generations to obtain mice with reduced adiponectin expression in both C57BL/6J and FVB/N backgrounds. The genotype was verified by PCR analysis of their genomic DNA using primers listed in Table 3. In addition, serum adiponectin levels were monitored using an in-house ELISA, with the standard curve generated from known concentrations of recombinant adiponectin. Note that mice with the genotype of PyVT(+/2)/ADN(2/2) (transgenic PyVT with adiponectin alleles) could not be found in all generations of alive litters, which included over 800 mice. On the other hand, their embryos were found to be dead at the early stage of foetal development. As a consequence, the sizes of litters with abnormal adiponectin expressions (3–5) were consistently smaller when compared to those of control PyVT breeding pairs (8–10). Therefore, the PyVT transgenic mice with adiponectin deficiency were referred to those with PyVT(+/2)/ADN(+/2) genotypes in this study. The circulating levels of adiponectin in PyVT(+/2)/ADN(+/2) FVB/N and C57BL/6J mice range from 3–15 mg/ml and 0.2– 5 mg/ml respectively, whereas PyVT(+/2)/ADN(+/+) mice in both FVB/N and C57BL/6J background have a much higher adiponectin level of over 20 mg/ml and 10 mg/ml respectively, with the median values increased by 4–5 folds. Tumor development was closely monitored every 2–3 days. Tumor latency was recorded as the age of mice when palpable tumors Adiponectin and Breast Cancer were first detected in at least one of the ten mammary fat pads. Tumor sizes were measured using digital vernier calipers and tumor volume calculated using the formula [sagittal dimension (mm)6(cross dimension (mm)2] / 2 and expressed in mm 3 . All animal experimental protocols were approved by the Animal Ethics Committee at the University of Hong Kong and their care was in accord with the institution guidelines. Sandwich ELISA for murine adiponectin The anti-murine adiponectin monoclonal antibody was bioti- nylated with a kit from Pierce, and free biotin was removed by dialysis. The polyclonal anti-murine adiponectin antibody was diluted to a concentration of 2 mg/ml, added to each well of a microtiter plate, and incubated overnight at 4 uC. The coated plate was washed 3 times with PBS containing 1% bovine serum albumin and blocked with 100 ml of PBS containing 1% bovine serum albumin and 0.05% Tween for 2 h. Mouse serum was diluted 1:10000, and 100 ml of the diluted samples were applied to each well along with the standard, incubated at 37 uC for 1 h, washed 3 times with PBS-Tween, and then incubated with 100 ml of the biotinylated monoclonal antibody (2 mg/ml) for another 2 h. After washing 3 times, the wells were incubated with streptavidin-conjugated horseradish peroxidase for 60 min and subsequently reacted with tetramethylbenzidine reagent for 15 min. 100 ml of 2 M H2SO4 was added to each well to stop the reaction, and the absorbance at 450 nm was measured. The intra- and interassay coefficients of variance were determined by measuring five plasma samples in a total of six independent assays with duplicate determinations. Primary tumor cell isolation, culture and re-implantation Primary cell isolation was performed as described previously with slight modifications [39]. Briefly, aseptically collected tumors from PyVT mice were mechanically minced, passaged through a 100-mm sterile nylon cell strainer (BD Falcon) and suspended in serum-free high glucose DMEM. Cells were further dissociated by serial passaging through a syringe with 25-gauge needles. After brief centrifugation at 1,000 r.p.m for 5 minutes to remove dead cell debris and the low-density stromal cells, the cell pellets were resuspended for viable cell counting using 0.4% trypan blue. 106 of isolated primary tumor cells were implanted into the third right mammary fat pad of female athymic nu/nu mice (4–6 weeks) by intraductal injection. Tumor development was monitored every 3– 4 days using caliper measurements (in millimeters) in two perpendicular dimensions (length and width). Tumor volumes were calculated as described above. Co-immunoprecipitation and Western Blotting Isolated tumor tissues were homogenized in RIPA buffer [50 mM Tris-HCl, pH 7.4; 1 mM EDTA; 150 mM NaCl; 1% Nonidel P40; 1% Triton X-100; 0.5% deoxycholic acid sodium salt; 1 mM NaF; 1 mM sodium orthovanadate; and complete protease inhibitor cocktail (Roche Applied Science, IN)] on ice and centrifuged for 5 min at 14,000 r.p.m to remove large debris. Protein concentration of the supernatant was determined by a BCA Protein Reagent Kit (Pierce Biotechnology, Rockford, IL). Five hundred micrograms of the total cell lysates were firstly incubated with rabbit IgG for 30 minutes, pre-cleared with 50 ml of protein G-Sepharose beads (Pierce Biotechnology, Rockford, IL), and then incubated with two micrograms of either Anti-Trx1 or Anti-PTEN antibody overnight at 4uC. 50 ml of protein G- Sepharose beads was added and incubated for 2 hrs at 4uC. Beads bound with immune complexes were collected by centrifugation and washed twice prior to elution into 90 ml of buffer containing PLoS ONE | www.plosone.org 12 March 2009 | Volume 4 | Issue 3 | e4968