Cyclooxygenases and UCP1 Figure 1. Cold exposure induces COX1 and COX2 expression in iWAT and iBAT. Sv129 mice were warm-acclimated at 28–30uC for 6 days and then transferred to 4–6uC. Samples for cryosections, RNA and protein extractions were prepared from iBAT and iWAT after 2, 4 and 6 days at 4– 6uC. A–B. Representative COX1 (A) and COX2 (B) immunoreactivity in iWAT from mice kept at 28–30uC for 6 days and after 6 days of cold exposure. C. Proteins were isolated from warm-acclimated mice (lane 1) and after 2, 4 and 6 days of cold exposure. COX1 and COX2 expression were determined by Western blotting. D. RNA was harvested from iBAT and iWAT from individual mice (n = 4 in each group) that were warm-acclimated or cold- exposed for 6 days. Expressions of genes were measured by RT-qPCR in duplicates and normalized to TBP (TATA box binding protein). The bars represent mean 6 standard error. * indicates statistical difference (p,0.05) compared to expression in warm acclimated mice. doi:10.1371/journal.pone.0011391.g001 acclimated mice with the COX inhibitor indomethacin and transferred the mice to 4uC. Measurements of rectal temperature revealed that mice treated with indomethacin had slightly, but significantly lower body temperature (Figure 3B). As expected, UCP1 expression was induced in both iBAT and iWAT in vehicle-treated mice (Figure 3C and D). While indomethacin treatment only slightly attenuated cold-induced UCP1 expression in iBAT, it almost completely prevented the induction of UCP1 expression in iWAT (Figure 3C and D). Thus, COX activity appeared to be necessary for cold-induced UCP1 expression in iWAT, but not in iBAT. In addition, indomethacin treatment attenuated cold-induced enhancement of PGC1a, Dio2, Cox8b, Eva1 and Cidea expression in iWAT, while preventing cold- induced repression of RIP140 and 4E-BP1 expression in iWAT (Figure 3D). Forced expression of COX2 induces UCP1 expression in Rb2/2 adipocytes Since indomethacin attenuated b-adrenergically stimulated UCP1 expression in Rb2/2 adipocytes and primary inguinal PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e11391