littermates from multiple litters over more than 12 months. Comprehensive data was collected for food consumption (Figure S1A) and body weight (BW) gain over this period of time (Figure 1B). A second phase of the study was started when an increased proportion of Cyp1b1-ko mice demonstrated DIO. Cyp1b1-ko mice that demonstrated DIO, which was characteristic of WT mice, were re-derived and refreshed by a single mating with WT animals. This Cyp1b1-ko colony predominantly retained high obesity with the HFD challenge. Subsequent back-crossing was completed by mating Cyp1b1-heterozygote females to Jackson WT males. The F3-F5 generations of this back-cross demonstrated obesity suppression and were used for glucose measurements. Additional mating examined the effect of breeding the smallest (S1&2) and largest (L1&2) progeny, respectively, from the initial backcross, as well as the effect of mating a heterozygous male or female, respectively, with a Cyp1b1-ko partner. Mice were sacrificed by CO2 asphyxiation and blood was collected by cardiac puncture. Tissues were weighed and flash frozen in liquid nitrogen and stored at −80° C for protein and mRNA analyses. Tissue samples were also frozen in sectioning medium or fixed in 10% formalin solution for histology.
