the mice had free access to water. For high fat diet challenge, normal chow was substituted by 60 kcal% fat diet (Research Diets, Inc. D12492). Body composition and bone mineral density were measured with Lunar PIXImus dual energy x-ray absorptiometry (DEXA) densitometry following the standard protocol. Measurement of the rates of metabolism and food consumption The metabolic rates of mice were measured by indirect caloriometry using the Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments). The food intake was also monitored by the system. 16,17-week-old mice were housed individually in the monitoring cage with free access to water and food for 3 days for acclimatization. Then the lean mass of the mice was measured by MRI (EchoMRI), before the animals were put back into the monitoring cages and monitored for the next 3 days for oxygen consumption, carbon dioxide production and food intake. IGF-1 ELISA Blood was collected from 4-week-old mice by cardiac puncturing. The sera were aliquoted and snap-frozen in liquid nitrogen. Before ELISA, the sera were extracted by acid-ethanol with cryo-precipitation to release IGF-1 from its binding proteins [51]. IGF-1 ELISA was performed using commercial kits (Signosis EA-2204, and AssayPro EMI1001-1) according to the manufac- turers’ protocols. The results were presented as the read-out from the microplate reader (spectrometer). Statistic analysis All statistic analyses were performed by one-way ANOVA (three sample sets) or unpaired t-test (two sample sets). ANCOVA was used when indicated. Supporting Information Figure S1 Generation and characterization of Fto gene-trap mice. (A) Schematic representation of Fto gene-trap strategy. (B) Schematic representation of predicted Fto wildtype and gene-trap coding sequence (CDS). (C) Western blot analysis of tissues from mice of all genotypes. (D) Growth curves of Fto+/+ and Ftogt/gt mice. For each genotype (Fto+/+/Ftogt/gt), n = 16/15 (males); 12/16 (females). All values are mean 6 s.e.m. (E) The body length of adult Fto+/+ and Ftogt/gt mice. At the time of the measurement, males were 13,14.5-week-old, and females 13,16-week-old. For each genotype (Fto+/+/Ftogt/gt,), n = 10/9 (males); 7/7 (females). All values are mean 6 s.e.m. (F) Body composition (fat mass/total tissue mass %) of Fto+/+ and Ftogt/gt mice fed on normal chow or high fat diet. Body composition was measured by DEXA (dual energy X-ray absorptiometry). For normal chow group, males were 13,14.5-week-old, and females 13,16-week-old. For each genotype (Fto+/+/Ftogt/gt,), n = 10/9 (males); 7/7 (females). For the high fat diet group, at the time of measurement, the mice had been fed on high fat diet (60 kcal % fat) for 12 weeks from 6-week-old. References Fto and Postnatal Growth For each genotype (Fto+/+/Ftogt/gt,), n = 11/6 (males); 8/8 (females). Statistical analyses were performed by unpaired t-test. All values are mean 6 s.e.m. Found at: doi:10.1371/journal.pone.0014005.s001 (0.74 MB TIF) Figure S2 Dermatitis in Fto knockout mice after high fat diet regimen. (A) A representative picture of an FtoD/D mice suffering dermatitis around the neck area. (B) A representative picture of Fto+/+ and FtoD/D mice after the high fat diet. The asterisk denotes the one with dermatitis. Found at: doi:10.1371/journal.pone.0014005.s002 (0.74 MB TIF) Figure S3 Metabolic rate of Fto mutant mice analyzed by ANCOVA. (A, C) Average hourly O2 consumption (A) and average hourly CO2 production (C) in relation to lean mass of 16,17-week-old male Fto+/D and FtoD/D mice during light and dark period. (B, D) Average hourly O2 consumption (B) and average hourly CO2 production (D) of 16,17-week-old male Fto+/D and FtoD/D mice adjusted by ANCOVA using an average lean mass. In (A)–(D), n = 7/7 (Fto+/D/FtoD/D). In (B) and (D), statistical analyses were performed by unpaired t-test using adjusted data. **P,0.01. All values are mean 6 s.e.m. (E, F) Average hourly O2 consumption (E) and CO2 production (F) in relation to lean mass of 16,17-week-old male FtoN+ and FtoND mice during light and dark period. n = 7/9 (FtoN+/FtoND). No significant linear relationship was detected. Found at: doi:10.1371/journal.pone.0014005.s003 (0.50 MB TIF) Table S1 Genotypes of 10,14-day-old pups from heterozygote intercrosses. Found at: doi:10.1371/journal.pone.0014005.s004 (0.04 MB PDF) Table S2 Genotypes of 1,3-day-old pups from heterozygote intercrosses that were found dead or missing. Found at: doi:10.1371/journal.pone.0014005.s005 (0.03 MB PDF) Table S3 Genotypes of E14.5,18.5 embryos from heterozygote intercrosses. Found at: doi:10.1371/journal.pone.0014005.s006 (0.03 MB PDF) Table S4 Number of mice that developed dermatitis on high fat diet. Found at: doi:10.1371/journal.pone.0014005.s007 (0.04 MB PDF) Acknowledgments We thank Drs. B. Lee, L. Chan, J. Qin, L. Chen, T. Yang, W. Chen, J. Mao, H. Zheng of BCM for discussion and sharing of reagents. We thank Dr. Y. Chen of BCM Nutrition Center for technical help. Author Contributions Conceived and designed the experiments: XG YHS QT PZ. Performed the experiments: XG YHS ML FW. Analyzed the data: XG. Wrote the paper: XG PZ. 1. Peters T, Ausmeier K, Ruther U (1999) Cloning of Fatso (Fto), a novel gene deleted by the Fused toes (Ft) mouse mutation. Mamm Genome 10: 983–986. 2. Dina C, Meyre D, Gallina S, Durand E, Korner A, et al. (2007) Variation in FTO contributes to childhood obesity and severe adult obesity. Nat Genet 39: 724–726. 3. Frayling TM, Timpson NJ, Weedon MN, Zeggini E, Freathy RM, et al. (2007) A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science 316: 889–894. 4. Scuteri A, Sanna S, Chen WM, Uda M, Albai G, et al. (2007) Genome-wide association scan shows genetic variants in the FTO gene are associated with obesity-related traits. PLoS Genet 3: e115. 5. Hinney A, Nguyen TT, Scherag A, Friedel S, Bronner G, et al. (2007) Genome wide association (GWA) study for early onset extreme obesity supports the role of fat mass and obesity associated gene (FTO) variants. PLoS ONE 2: e1361. 6. Ohashi J, Naka I, Kimura R, Natsuhara K, Yamauchi T, et al. (2007) FTO polymorphisms in oceanic populations. J Hum Genet 52: 1031–1035. PLoS ONE | www.plosone.org 12 November 2010 | Volume 5 | Issue 11 | e14005