(A) Schematic representation of the Hdac11-floxed allele (Hdac11^fl), with loxP sites flanking the 162 base pair (bp) exon 2; the predicted size of the resulting PCR product generated from genomic DNA and forward (F) and reverse (R) primers is shown. (B) Schematic representation of the cross between adiponectin promoter–driven Cre recombinase (Adipoq-Cre) transgenic mice and Hdac11^fl/fl mice and the genomic DNA PCR approach to assessing excision of Hdac11 exon 2. (C) Genomic DNA PCR products from Hdac11^fl/fl mice without (–) and with (+) the Adipoq-Cre transgene. (D) Diagram describing the genotype and definition of the adipocyte-specific conditional Hdac11-KO (Hd11^cKO) and control Hd11^fl/fl mice. (E) Immunoblot analysis of HDAC11 and UCP1 protein in epididymal white adipose tissue (eWAT), inguinal white adipose tissue (ingWAT), and interscapular brown adipose tissue (BAT) from 10- to 12-week-old control and Hdac11^cKO mice. GAPDH served as a loading control; n = 3 biological replicates/group. (F and G) Densitometric analysis of HDAC11 and UCP1 protein in E, normalized to GAPDH and plotted relative to controls. Data are depicted as mean + SEM, with *P < 0.05 as determined by 2-tailed, unpaired t test. (H) Schematic representation of the cell culture experiment with recombinant Cre recombinase (recCre). (I) Immunoblot analysis of HDAC11 and UCP1 protein in preadipocytes. (J) Schematic representation of the 24-hour 4°C challenge experiment. (K) Core body temperature over time. Data are depicted as mean ± SEM, with *P < 0.05 vs. control mice at a given time as determined by 2-way ANOVA with Šidák’s multiple-comparison test; n = 7 biological replicates/group. (L) Adipose tissue weight, normalized to tibia length, determined after the 24-hour 4°C challenge. Data are depicted as mean + SEM, with *P < 0.05 as determined by 2-tailed, unpaired t test. (M) Immunohistochemistry of UCP1 protein in eWAT from mice sacrificed following the 24-hour 4°C challenge. Scale bars: 200 μm.