A. Nutritional regulation of Irf4 mRNA expression in murine white adipose tissue (WAT). Male FVB mice were sacrificed in the fed state, after a 24hr fast, or 24hr after refeeding (n=7-8). Data are normalized to 36B4 expression and are expressed as fold induction relative to Irf4 mRNA in the fed state. Results expressed as mean ± SD. B. Nutritional regulation of IRF4 protein expression in murine WAT, brown adipose tissue (BAT), and spleen. C. IRF4 mRNA expression in human WAT, sampled pre and post-fasting (n=7). *p<0.05 versus pre fasting. D. Regulation of Irf4 mRNA expression by insulin in 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated in serum-free DMEM with insulin at the indicated doses for 8hrs (n=3). Data are normalized to 36B4 and presented as expression relative to 0nM insulin. *p<0.05 versus 0nM insulin. Results expressed as mean ± SD. E. Irf4 mRNA expression in WAT of streptozotocin (STZ)-treated mice. Irf4 mRNA expression was measured by Q-PCR in WAT of 10-week-old male mice given vehicle (control), STZ, or STZ followed by insulin replacement for 24h (STZ+Ins) (n=8-10/group). Data are normalized to 36B4 and presented as expression relative to control, *p<0.05. Results expressed as mean ± SD. F. Irf4 mRNA expression in WAT of Fat Insulin Receptor Knockout (FIRKO) mice (n=4/group). Data are normalized to 36B4 and presented as expression relative to control. Results expressed as mean ± SD. *p<0.05 versus control. G. Irf4 mRNA expression in 3T3-L1ΔCAR adipocytes transduced with adenovirus expressing EGFP, wild type (WT), constitutively active (CA), or dominant negative (DN) FoxO1. Data are normalized to 36B4 and presented as fold induction relative to EGFP cells. *p<0.05 versus EGFP cells. Results expressed as mean ± SD. H. Luciferase activity of Irf4 promoter constructs in 3T3-L1 adipocytes. Five days after adipogenic stimulation, 3T3-L1 cells were transfected with the indicated Irf4 promoter construct. Luciferase activity was measured 24 hr after transfection in the presence (filled bar) or absence (open bar) of insulin. Results are expressed as mean ± SD (n = 3). *p < 0.05 relative to the same construct without insulin. Results expressed as mean ± SD. I. ChIP assay of FoxO1 binding to Irf4 promoter constructs in 3T3-L1 adipocytes in the presence (filled bar) or absence (open bar) of insulin (n=3). All results normalized to IgG without insulin, and expressed as mean ± SD.