Differentiation of 3T3-L1 Preadipocytes

3T3-L1 preadipocytes were maintained in DMEM containing 10% calf serum (Hyclone Laboratories, South Logan, UT). Two day postconfluent 3T3-L1 cells (designated day 0) were induced to differentiate by addition of a standard cocktail composed of 0.5 mM IBMX, 1 μM Dex, and 10 μg/ml insulin in 10% FBS for 2 days (designated as MDI cocktail). The cells were then cultured in DMEM supplemented with 10% FBS and 5 μg/ml insulin. The medium was replaced with fresh medium every 2 days. For the AG490 experiments, cells were preincubated with 10 μM AG490 for 1 h and then treated with 10 ng/ml IL-4. For cell counting, cells were harvested and stained with trypan blue (0.5%; Biological Industries, Kibbutz Beit Haemek, Israel), and viable cells were counted with the use of a hemocytometer (Lauda-Königshofen, Marienfeld-Superior, Germany).