performed in duplicates as described sequences are available on request. Western blotting Preparation of extracts from mouse tissues or whole cell dishes, electrophoresis, blotting, visualization and stripping of membranes were performed as described [84]. Primary antibodies used were goat anti-COX1, goat anti-COX2, rabbit anti-UCP1 and rabbit anti-TFIIB antibodies (Santa Cruz Biotechnology). Secondary antibodies were horseradish peroxidase-conjugated anti-mouse, anti-goat or anti-rabbit antibodies obtained from DAKO. Immunohistochemistry COX1 (M-20; sc 1754) and COX2 (M-19; sc 1747) antibodies were obtained from Santa Cruz Biotechnology, diluted 1:300 on cryosections and 1:100 on paraffin-embedded sections (for COX1). Lung [85] and skin [86] were used as positive control for both COX1 and COX2 antibodies. Histological analyses Parts of adipose tissue were fixed in 4% formaldehyde in PB buffer for 24 h, washed in PB, dehydrated in ethanol, embedded in paraffin after 2610 min xylen treatment. Sections (8 mm thick) of the embedded tissue sections were subjected to standard hematoxylin and eosin staining. Supporting Information Text S1 Experimental. References 1. Cannon B, Nedergaard J (2004) Brown Adipose Tissue: Function and Physiological Significance. Physiol Rev 84: 277–359. 2. Kopecky J, Hodny Z, Rossmeisl M, Syrovy I, Kozak LP (1996) Reduction of dietary obesity in aP2-Ucp transgenic mice: physiology and adipose tissue distribution. Am J Physiol 270: E768–E775. 3. Kopecky J, Rossmeisl M, Hodny Z, Syrovy I, Horakova M, et al. (1996) Reduction of dietary obesity in aP2-Ucp transgenic mice: mechanism and adipose tissue morphology. Am J Physiol Endocrinol Metab 270: E776–E786. 4. Cederberg A, Gronning LM, Ahren B, Tasken K, Carlsson P, et al. 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Cannon B, Nedergaard J (2004) Brown Adipose Tissue: Function and Physiological Significance. Physiol Rev 84: 277–359. earlier [83]. Primer at: doi:10.1371/journal.pone.0011391.s001 (0.04 MB PLoS ONE | www.plosone.org 11 June 2010 | Volume 5 | Issue 6 | e11391 Cyclooxygenases and UCP1 Found DOC) Figure S1 COX1 and COX2 are mainly expressed in the stromal-vascular fraction of iBAT in warm-acclimated mice. A. Sv129 mice were warm-acclimated at 28–30uC for 6 days and then transferred to 4–6uC. Samples for cryosections were prepared from iBAT and iWAT after four days of cold exposure. Representative COX2 immunoblots in iBAT B. iBAT from warm-acclimated mice was fractionated into SVF and adipocyte fractions, respectively. Expression levels of COX1 and COX2 were determined by RT-qPCR and Western blotting. Found at: doi:10.1371/journal.pone.0011391.s002 (15.01 MB EPS) Figure S2 Inhibition of COX does not prevent isoproterenol/9- cis-retinoic acid-induced UCP1 expression in WT-1 cells. Differentiated WT-1 cells were treated with a combination of isoproterenol (100 nM) and 9-cis-retinoic acid (1 mM) for 24 h. When included, indomethacin (1 mM) were added 2 h before isoproterenol and 9-cis retinoic acid. UCP1 and PGE1a expressions were determined by RT-qPCR. Found at: doi:10.1371/journal.pone.0011391.s003 (2.44 MB EPS) Author Contributions Conceived and designed the experiments: LM KK. Performed the experiments: LM LMP HHL EF IB QH RKP PH TM RDM PA JM MLB JBH BC JN JW SC PV SOD. Analyzed the data: LM LMP HHL EF RKP PH TM PA JM MLB JBH BC JN JW SC PV SOD KK. Wrote the paper: LM LMP BC JN SOD KK. 15. Cypess AM, Lehman S, Williams G, Tal I, Rodman D, et al. (2009) Identification and Importance of Brown Adipose Tissue in Adult Humans. N Engl J Med 360: 1509–1517. 16. van Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM, Drossaerts JMAF, Kemerink GJ, et al. (2009) Cold-Activated Brown Adipose Tissue in Healthy Men. N Engl J Med 360: 1500–1508. 17. 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