of 10 nM fluorescein (Bio-Rad, Hercules, CA) in a final volume of 25 ml. Program setting for mouse samples were as follows: 2 min at 50uC, 2 min at 95uC, 40 cycles of 15 sec at 95uC and 30 sec at 62–64uC (depending on primer pair). Program setting for human samples were as follows: 10 min at 95uC, 40 cycles of 15 sec at 95uC 20 sec at 63uC. For primer information see Table S1. Primer pairs were optimized prior to use for primer concentration and annealing temperature to achieve PCR amplification efficiency between 95–105%. Mouse samples were normalized towards b- actin and human samples towards GAPDH. Relative quantifica- tion of mRNA was calculated using the ‘‘Comparative Ct method’’ as described in User Bulletin 2 from Applied Biosystems. For mice, statistical analysis was carried out using Mann-Whitney U test and for humans using ANOVA (Table S5). Measurement of TRAP enzyme activity TRAP enzyme activity was measured [68] in tissue homoge- nates prepared as follows. Adipose tissue from male and female mice (WT; n = 11, TRAP+; n = 10, TRAP+p; n = 10) was homogenized in 0.15 M KCl+0.1% Triton X-100+Pefabloc (10 mg/ml)+Complete, Protease Inhibitor Cocktail Tablets (1 tablet/50 ml solution) (Boehringer Mannheim, Mannheim, Ger- many) and centrifuged at 3200 6 g for 30 minutes. The supernatant was then assayed for TRAP enzyme activity. Human studies Human abdominal subcutaneous adipose tissue was obtained from different sources. For the isolation of pre-adipocytes and mesenchymal stem cells (MSC), tissue was obtained as the product of cosmetic liposuction on otherwise healthy non-obese women (body mass index ( = BMI): 22–36 kg/m2; age 22–56 years; n = 10). For immunohistochemistry, tissue was obtained during surgery from three obese subjects undergoing gastric banding. For the comparison of gene and protein expression in lean versus obese subjects a frozen (270uC) 300 mg tissue piece was used. It was obtained by needle biopsy from lean (BMI,25 kg/m2) or obese (BMI.30 kg/m2) but otherwise healthy women participating in ongoing studies of the genetic regulation of human fat cell function. For those in the gene expression study age (mean6SD) was 36614 and 3967 years in lean (n = 14) and obese (n = 14) subjects, respectively. BMI was 2362 and 3664 kg/m2 respectively. Fat cell volume was 450 6140 and 8226240 pL in lean and obese, respectively. The obese group in the gene expression study was sub-divided in two groups according to fat cell size; hyperplasia (fat cell volume of 6416450 pL; n = 7) and hypertrophia (cell volume of 10046490 pL; n = 7) with no between group difference in BMI. In the protein expression study age was 4064 years (lean (n = 7)) and 40613 years (obese (n = 12)), the BMI was 2361 kg/m2 and 3865 kg/m2, and fat cell volume was 4906190 pL and 8686223 pL, respectively. The obese group was sub-divided into a hyperplastic (fat cell volume 6596420 pL; n = 6) and hypertrophic (fat cell volume 10776570 pL; n = 6) group) with no between group difference in BMI. The human ethics committee at the Karolinska University Hospital Huddinge approved the study, which was explained in detail to each subject and written informed consent was obtained. Unpaired t-test was used to compare lean and obese subjects and adipose tissue versus adipocytes. ANOVA was used to compare lean subjects with the hypertrophic and hyperplastic groups. Degrees of freedom in these experiments were 1, 1 and 2, respectively. Electrophoresis and immunoblot analysis Partially purified [11] culture media from 16106 RAW 264.7 cells or 70mU TRAP from WT/TRAP+p/TRAP+ adipose tissue was subjected to SDS-PAGE using 12% NuPage gels (Invitrogen, TRAP Induces Obesity Carlsbad, CA) run with MOPS buffer and transferred to PVDF membranes (Bio-Rad, Hercules, CA). Membranes were blocked using 1% TBST (100 mM Tris-HCl pH 7.6, 154 mM NaCl, 1% Tween-20) and stained with rabbit anti-mouse monomeric TRAP [11] 1:1500 or rabbit anti-rat total TRAP 1:1500 [69], goat anti- rabbit HRP 1:10 000 (Calbiochem, La Jolla, CA) and developed using Renaissance (NEN Life Science, Boston, MA). Human samples; 100 mg of total protein total obtained from protein lysates was separated on 12% PAA-gels, transferred onto PVDF mem- branes and stained using rabbit anti-mouse monomeric TRAP or rabbit anti-rat total TRAP as described above. Bands were detected using SupersignalH (Pierce, Rockford, IL). Relative expression was determined using Chemidoc XRS System (Bio-Rad, Hercules, CA). Immunohistochemistry Immunohistochemistry was carried out mainly as previously described [11]. Mouse adipose tissue from male and female mice; paraffin sections (WT; n = 3, TRAP+; n = 3) were treated with 0.1% trypsin (Sigma-Aldrich, St. Louis, MO) at 37uC for 30 minutes and stained for macrophages using F4/80 monoclonal antibody (1:50) (Serotec, Oxford, UK). ChemMate Detection Kit Peroxidase/DAB rabbit/mouse (Dako, Glostrup, Denmark) was used as secondary antibodies and developing solution. Human adipose tissues; microwave treated (1mM EDTA pH 8 for 10 minutes at 900 W) paraffin sections were stained with rabbit anti-rat total TRAP 1:50 [69] recognizing both monomeric and the two-subunit TRAP or rabbit anti-mouse monomeric TRAP antibody 1:50 [11] and CD68 1:100 (Dako, Glostrup, Denmark). Secondary antibodies were ALEXA 568 goat anti-rabbit Fab2 1:250 and ALEXA 488 goat anti-mouse Fab2 fragments 1:100 (Invitrogen, Carlsbad, CA). Sections were then examined using a Leica TCS NT ArKr laser confocal microscope (Leica Micro- systems AG, Wetzler, Germany). Expression, purification and cleavage of recombinant rat TRAP Recombinant rat TRAP was expressed in Sf9 insect cells and purified as described [70]. To generate the proteolytically processed form, recombinant rat TRAP was digested at 37uC for 40 minutes with recombinant human cathepsin K (kindly provided by Dr. Robert Dodds) in 5mM NaAc pH 5.5, 1 mM EDTA and 10 mM DTT using a 1:1 molar ratio. Proliferation and differentiation of 3T3-L1 cells in the presence of TRAP For proliferation experiments (n = 4, df = 3); 3T3-L1 (ATCC (LGC Promochem, Bora˚s, Sweden)) cells (2,000 cells/cm2) were cultured in DMEM/F12 Glutamax II, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, penicillin/streptomycin, 2.5% calf bovine serum +/2 cleaved (600 U/mg) or monomeric (50 U/mg) TRAP (1029 M–10212 M). After 24, 48 and 72 h, cells were labeled for 2 h with BrdU, fixed and BrdU incorporation was measured using Cell Proliferation ELISA, BrdU kit (Roche, Mannheim, Germany). For differentiation experiments (n = 4, df = 3); 3T3-L1 cells 2 (6,000 cells/cm ) were grown into confluence in DMEM/F12 Glutamax II, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, penicillin/streptomycin, 10% calf bovine serum +/2 cleaved or monomeric TRAP (1029 M–10212 M)). At 2–3 days after conflu- ence, 0.5 mM isobutylmethylxanthine, 1 mM dexamethasone and 10 mg/ml bovine insulin was added to the media to start differentiation. Cells were then cultured +/2 roziglitazone (1 mM), cleaved (600 U/mg) or monomeric (50 U/mg) TRAP (1029 M–10212 M). After 48 h, dexamethasone, isobutylmethyl- PLoS ONE | www.plosone.org 11 March 2008 | Volume 3 | Issue 3 | e1713