protease and phosphatase inhibitor cocktail (Sigma) by 20 strokes in a glass-glass Potter apparatus at 4uC. The homogenates were centrifuged at 20,0006g for 30 min at 4uC, and the supernatant was used as whole cell extracts. Gel mobility shift assays were performed essentially as described in [49]. Purified oligonucleotides were annealed and labeled with [32P]-dCTP (GE Healthcare) using the Klenow fragment of DNA polymerase (Life Technologies, Inc., Paisley, UK) to a specific activity of approximately 108 cpm/mg of DNA. Unlabeled oligonucleotides were used as competitors. Oligonucleotide sequences were as follows: IGF-2-forward: 59-GCCAGATGTTCTTGGAATGGGACA-39; IGF-2-rev: 59- GTGTCCCATTCCAAGAACATCTGG-39. Protein-DNA com- plexes were separated from free DNA by electrophoresis on nondenaturing 4.5% polyacrylamide gel in 0.25X Tris-borate- EDTA buffer at 200 V for 1 h. Gels were dried and exposed to x-ray film at 280uC. Proliferation assays T37i cells were cultured in 24-well plates. Post-confluent cells (d2) and differentiated cells (d7) were rinsed and grown for 12 h in a serum-free medium (DMEM/HAM’s F12 containing 2 nM T3), then maintained at 37uC in the same medium for an additional 8 h period, in the absence or presence of 20 nM IGF-2 or insulin, and 1 mCi/well of [3H]-Thymidine (GE Healthcare) was added during the last 2 h. Cells were washed twice with ice-cold PBS and lysed in 1% SDS. DNA was precipitated by 10% trichloroacetic acid. The resulting trichloroacetic acid-insoluble pellet was washed two times with 5% trichloroacetic acid and collected on Whatman GF/C filters. [3H]-Thymidine incorporation into DNA was measured by scintillation counting. Statistical analyses All data were prepared for analysis with standard spread sheet software (Microsoft Excel). Data are expressed as the mean6SE. Statistical analysis was performed using Student’s t test to determine significant differences among groups and were performed using the computer software Prism 4 (GraphPad Software, San Diego, CA). We performed ANOVA, if statistical significance (P values *,0.05, **,0.01 and ***,0.001) was achieved and post-test analysis to account for multiple compar- isons was done. SUPPORTING INFORMATION Figure S1 Lipogenic capacity of brown adipocytes is not impaired in PRLR KO mice. A) Citrate synthase activity (CS) and B) Glycerol-3-phosphate dehydrogenase (G3PDH) were REFERENCES 1. Goffin V, Binart N, Touraine P, Kelly PA (2002) Prolactin: the new biology of an old hormone. Annu Rev Physiol 64: 47–67. 2. Bole-Feysot C, Goffin V, Edery M, Binart N, Kelly PA (1998) Prolactin and its receptor: actions, signal transduction pathways and phenotypes observed in prolactin receptor knockout mice. Endocr Rev 19: 225–268. 3. LeBaron MJ, Ahonen TJ, Nevalainen MT, Rui H (2007) In vivo response-based identification of direct hormone target cell populations using high-density tissue arrays. Endocrinology 148: 989–1008. 4. Freemark M, Kirk K, Pihoker C, Robertson MC, Shiu RC, et al. (1993) Pregnancy lactogens in the rat conceptus and fetus: circulating levels, distribution of binding, and expression of receptor messenger RNA. Endocri- nology 133: 1830–1842. 5. Royster M, Driscoll P, Kelly PA, Freemark M (1995) The prolactin receptor in the fetal rat: cellular localization of messenger RNA, immunoreactive protein, and ligand binding activity and induction of expression in late gestation. Endocrinology 136: 3892–3900. Prolactin and Brown Adipocytes determined in homogenates isolated from the BAT of WT and KO mice. Results represent mean6SE of 6 animals. NS: not significant Found at: doi:10.1371/journal.pone.0001535.s001 (0.18 MB TIF) Figure S2 COX activity nor COX2 DNA content are altered in PRLR KO mice. A) Mitochondrial COX2 DNA content was determined by quantitative real time PCR in homogenates isolated from the BAT of WT and KO mice. Results, expressed as attomol of COX2/fmol of 18S, represent mean6SE of 6 animals. NS: not significant B) Cytochrome c oxidase acitivity (COX, Fig. S2A) was determined in homogenates isolated from the BAT of WT and KO mice. Results represent mean6SE of 6 animals. NS: not significant. Found at: doi:10.1371/journal.pone.0001535.s002 (0.17 MB TIF) Figure S3 Differentiated T37i brown adipocytes expressed IGF- 1R, IGF-2R and IR-A. RT-PCR analyses of IGF-1 receptor (IGF- 1R), mannose-6 phosphate receptor (IGF-2R), and Insulin receptor form A (IR-A) in fully differentiated T37i cells in the presence (+) or the absence (-) of the reverse transcriptase. Oligonucleotides were as followed: IGF-1R-forward 59-CGGTGACTTCTGCTCAAAT- GC-39; IGF-1R-reverse 59-GAATGGCGGATCTTCACGTAG- 39. IGF-2R-forward 59-CGAGGCCGAAACTCAGATAGA-39; IGF-2R-reverse 59-AAAACGGATGATGAATGCTGTG-39; IR- A-forward 59-GCTGGACTGTGGTGGATATTGA-39; IR-A-re- verse 59-TCAAGGGATCTTCGCTTTCG-39. Found at: doi:10.1371/journal.pone.0001535.s003 (0.15 MB TIF) at: doi:10.1371/journal.pone.0001535.s004 (0.03 MB Text S1 Found DOC) ACKNOWLEDGMENTS The authors are indebted to Franc¸oise Cluzeaud (CRB3, Inserm U773, Medical School Bichat, Paris) for her assistance in electron-microscopy, to Hannia Bouzinba-Segard for her help during initial immunoprecipitation experiments, to Dr. S. Mohan (Loma Linda University) for measurement of plasma IGF-2, to Pr. D. Ricquier (CNRS UPR 9078, Paris) for providing UCP1 antibody and to Dr. Anne Lombe`s (Inserm U582, Salpe´trie`re hospital, Paris) for critical and helpful discussions. The assistance of Annick Ganieux (IFR 93 Biceˆtre) for the preparation of plasmids and Ge´rard Pivert for histological procedures are also gratefully acknowledged. Author Contributions Conceived and designed the experiments: BF NB SV ML. Performed the experiments: BF SV NS ML. Analyzed the data: BF NB SV NS MF ML. Wrote the paper: NB SV MF ML. 6. Freemark M, Driscoll P, Maaskant R, Petryk A, Kelly PA (1997) Ontogenesis of prolactin receptors in the human fetus in early gestation. Implications for tissue differentiation and development. J Clin Invest 99: 1107–1117. 7. Cannon B, Nedergaard J (2004) Brown adipose tissue: function and physiological significance. Physiol Rev 84: 277–359. 8. Ricquier D, Bouillaud F (2000) Mitochondrial uncoupling proteins: from mitochondria to the regulation of energy balance. J Physiol 529 Pt 1: 3–10. 9. Handschin C, Spiegelman BM (2006) Peroxisome proliferator-activated receptor gamma coactivator 1 coactivators, energy homeostasis, and metabolism. Endocr Rev 27: 728–735. 10. Rosen ED, Spiegelman BM (2000) Molecular regulation of adipogenesis. Annu Rev Cell Dev Biol 16: 145–171. 11. Rosen ED, Hsu CH, Wang X, Sakai S, Freeman MW, et al. (2002) C/EBPalpha induces adipogenesis through PPARgamma: a unified pathway. Genes Dev 16: 22–26. PLoS ONE | www.plosone.org 12 February 2008 | Issue 2 | e1535