Animals. For immunohistochemical analyses, mice and rats were kept with free access to standard lab chow and water. The protocol for treatment with CL-316243 has been described (1). Before cold exposure, C57BL/6 mice (10 weeks old) were preacclimated to 30°C for 2 weeks and then transferred to 4°C.

Cell Culture, Transduction, and Differentiation. For the differentiation of all cell types, including those transduced with retrovirus, cells were grown to confluence and induced to differentiate in medium containing dexamethasone (1 m M) (Sigma), methylisobutylxanthine (0.5 mM) (Sigma), insulin (5 m g/ml), and rosiglitazone (1 m M). After 48 h, the induction medium was replaced by medium containing insulin and rosiglitazone. Medium was refreshed every other day. For Figs. 2, 4A, and 7B, mouse embryo fibroblasts (MEFs) at the indicated days of differentiation were harvested for either RNA isolation or protein extraction. The cells used in Fig. 7A were deprived of rosiglitazone from days 8 to 10, after which they were induced for 10 h with the substances indicated in Fig. 7A. Forskolin, isoproterenol, 9-cis-retinoic acid, and BRL37344 were obtained from Sigma. All cells used in Fig. 1E were differentiated as described above and stimulated with 9-cis-retinoic acid (1 m M) and isoproterenol (100 nM) from days 8 to 10. Suppression of cAMP-dependent protein kinase (PKA) activity during the differentiation of Rb^-/- MEFs (Figs. 4D and 8) was achieved by the addition of H-89 (Sigma) to the medium (10 m M). H-89 was added 2 h before the addition of induction medium and refreshed at all subsequent medium changes. Control cells received similar amounts of vehicle (0.1% DMSO).

Retroviral Vectors. pBabe-SV40 (simian virus 40) TAg and pBabe-TAg-K1 retroviral vectors were kindly provided by James A. DeCaprio (Dana-Farber Cancer Institute, Boston). TAg-K1 contains a point-mutation (Glu-107® Lys) in the Leu-Phe-Cys-Ser-Glu pRB interaction motif.

Antibodies. The following antibodies were used: Anti-UCP-1 (AB3038) (UCP, uncoupling protein) from Chemicon, anti-SV40 TAg (sc-147) and anti-CDK4 (sc-260) from Santa Cruz Biotechnology, anti-CREB (no. 06-863) and anti-phospho-(Ser 133)-CREB (no. 05-677) from Upstate Biotechnology, anti-pRB (G3-245) from BD PharMingen, anti-phospho-(Ser 780)-pRB (no. 9307) from Cell Signaling Technology, anti-actin (A2066) from Sigma, and horseradish peroxidase-conjugated anti-rabbit and anti-mouse from DAKO.

Immunohistochemistry and Morphometry. C57BL/6 mice and Sprague--Dawley rats were used for immunohistochemical studies. Tissues were isolated from mouse embryos on embryonic days 19-20 (E19-20), and during the early neonatal period on days 6-10. Interscapular brown and epididymal white adipose depots were dissected and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Intestine, skin, and testis were sampled as controls. The retroperitoneal fat depot of Sprague--Dawley rats (20 weeks of age) treated with 1 mg/kg/day of CL-316243 (n = 3) or saline (n = 3) for 7 days (1) were also examined. Sections were incubated with 3% H₂O₂ in water for 10 min at room temperature to block endogenous peroxide activity and incubated in 1:75 normal horse serum in PBS for 20 min at room temperature to block nonspecific binding. Sections were incubated with the anti-pRB antibody (G3-245, BD PharMingen), diluted 1:10 overnight at 4°C, then with biotinylated anti-mouse IgG diluted 1:200 in PBS for 30 min and finally with the avidin--biotin complex conjugated with horseradish peroxidase (Vectastain ABC Kit, Vector Laboratories). Peroxidase activity was revealed by diaminobenzidine. Sections were counterstained with hematoxylin, dehydrated, and finally mounted in Eukitt. Tests of the specificity of reaction were performed by omitting the primary antibody and by substitution with mouse IgG as the primary antibody. Microwave antigen retrieval [double irradiation in a 650-W microwave oven for 5 min in 0.01 M citrate buffer (pH 6.0), with 5 min cooling between the first and second irradiation] was used. Morphometric quantitations were performed at the level of light microscopy on retroperitoneal white adipose tissue sections of saline- or CL-316243-treated animals. The percentages of nuclei stained by the anti-pRB antibody were counted at high magnification (oil immersion with the ´ 100 objective). One midline sagittal section of the entire depot on one side of each of the three animals was used. The whole field of the section was used to count 100-120 unilocular and multilocular (the latter only in CL-316243-treated animals) adipocytes. The data are expressed as mean ± SD. Statistical analysis was performed using Student’s t test.

1. Himms-Hagen, J., Melnyk, A., Zingaretti, M. C., Ceresi, E., Barbatelli, G. & Cinti, S. (2000) Am. J. Physiol. 279, C670–C681.