xanthine and roziglitazone were omitted. After an additional 48h (i.e. 4 days after the start of differentiation), cells were lysed and GPDH activity was measured [71]. Statistical analysis was performed using t-test. Proliferation and differentiation of human mesenchymal stem cells derived from adipose tissue in presence of TRAP Human MSC were isolated from a subcutaneous lipoaspirate, grown to 60–70% confluence and passage for at least 20 passages, as described [72]. For proliferation experiments (n = 3, df = 2); 2 hMSC (passage 9) (2,000 cells/cm ) were cultured in DMEM Glutamax I, 1 g/L glucose, penicillin/streptomycin, 2.5% fetal bovine serum in the presence or absence of cleaved (600U/mg) or monomeric (50U/mg) TRAP (1029 M–10212 M). After 48 h, cells were labeled for 2h with BrdU, fixed and BrdU incorporation was measured as described above. For differentiation experiments (n = 3, df = 2); hMSC (passage 18) were differentiated as described [72] in the presence or absence of cleaved or monomeric TRAP (1029 M). Twelve days after the start of differentiation cells were lysed and GPDH activity was measured [71]. Statistical analysis was performed using t-test. Differentiation and proliferation of preadipocytes isolated from human adipose tissue in presence of TRAP. Isolation of pre-adipocytes were performed as previously described [73]. For proliferation experiments (n = 3, df = 2); cells (4000 cells/cm2) were cultured in growth medium containing 2.5% fetal bovine serum in the presence or absence of cleaved or monomeric TRAP (1029 M–10212 M). After 5 days, cells were labelled for 2 h with BrdU, fixed and BrdU incorporation was measured as described above. For differentiation experiments (n = 3, df = 2); cells were differentiated as described [74] in the presence or absence of cleaved (600U/mg) or monomeric (50 U/ mg) TRAP (1029 M–10212 M). Twelve days after the start of differentiation, cells were lysed in GPDH buffer and GPDH activity was measured as described [71]. Statistical analysis was performed using t-test. Secretion of TRAP from macrophages RAW 264.7 cells were cultured in a 37uC humidified 5% CO2 atmosphere in DMEM media (Gibco, St Louis, MO) supplement- ed with 10% FCS (Gibco, St Louis, MO) and 0.1 mg/ml gentamycin (Gibco, St Louis, MO). For stimulation, 0.256106 RAW 264.7 cells/ml (passage 3) was treated with IFNc (500 U/ ml) (Invitrogen, Carlsbad, CA) for 16 h., and then with LPS (1 ng/ml) (Sigma-Aldrich, St. Louis, MO) for an additional 24 h. Metabolic studies on the TRAP over expressing mice Lipolysis and lipogenesis experiments were conducted on isolated adipocytes as described [73] on fat (pooled fractions of gonadal, mesenteric and inguinal fat from the same animal) from male and female mice (WT; n = 8 and TRAP+; n = 8). After a 2 h incubation, the cell suspension was subjected to measurement of active uptake into total fat cell lipids (lipogenesis index). Statistical analysis was performed using ANOVA. References TRAP Induces Obesity Measurement of glucose, insulin and HOMA index Glucose (WT; n = 3 and TRAP+; n = 3), HOMA index (WT; n = 3 and TRAP+; n = 3) and insulin (WT; n = 8 and TRAP+; n = 3) were determined individually on serum from male and female mice, using a Monarch automated analyzer (ILS Laboratories Scandinavia AB, Sollentuna, Sweden) or a RIA assay (Linco Research Inc., St. Charles, MO), respectively. Statistical analysis was performed using Mann-Whitney U test. Measurement of serum leptin, adiponectin and TNFa Serum levels of leptin in male and female mice .4 months of age (WT; n = 26 and TRAP+; n = 13) (Quantikine Mouse Leptin Immunoassay, R&D Systems, Inc., Minneapolis, MN), of adiponectin in male and female mice .4 months of age (WT; n = 11 and TRAP+; n = 7) (Adiponectin Mouse ELISA, BioVen- dor, Heidelberg, Germany) and of TNFa in male and female mice .4 months of age (WT; n = 8 and TRAP+; n = 5) (Ready-Set-Go! Mouse TNFa ELISA, ebioscience, San Diego, CA) was measured. Statistical analysis was carried out using Mann-Whitney U test. Supporting Information Table S1 Found at: doi:10.1371/journal.pone.0001713.s001 (0.05 DOC) Table S2 Found at: doi:10.1371/journal.pone.0001713.s002 (0.03 DOC) Table S3 Found at: doi:10.1371/journal.pone.0001713.s003 (0.06 DOC) Table S4 Found at: doi:10.1371/journal.pone.0001713.s004 (0.04 DOC) Table S5 Found at: doi:10.1371/journal.pone.0001713.s005 (0.03 DOC) Table S6 Found at: doi:10.1371/journal.pone.0001713.s006 (0.05 DOC) MB MB MB MB MB MB Acknowledgments The authors thank Dr Jussi Halleen for providing the breeding stocks of the WT and transgenic TRAP over expressing mice, Mrs Gaby A˚berg, Mrs Eva Sjo¨lin, Mrs Maria Norga˚rd and Mrs Jenny Verner-Carlsson for excellent technical assistance. Author Contributions Conceived and designed the experiments: GA PL Vv PA. Performed the experiments: PL Vv MR MK PP CC. Analyzed the data: GA PL Vv MR MK PP AC PA CC. Contributed reagents/materials/analysis tools: AC DH. Wrote the paper: GA PL Vv PA DH. 1. 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