Representative lines from each group were further analyzed functionally. Bat1, bat6, bat8 for brown fat cells; X9, X3, D16, D12 for beige fat cells and N13, X7, J6, G18 for white fat cells. All these lines presented similar levels of adipogenesis and fat cell specific markers were measured via qPCR. See . Similar results were observed in more than three independent experiments. (A) Total RNA was isolated from the differentiated brown, beige and white clonal cell lines and was assayed for mRNA levels of brown fat-like genes (Ucp-1, Cidea and Cox7a1) by qPCR. (B) Total RNA was isolated from the differentiated brown, beige and white clonal cell lines treated with 10μM forskolin for 4 hours and was assayed for mRNA levels of Ucp-1. (C) Protein lysates were isolated from the differentiated brown, beige and white clonal lines either unstimulated or treated with 10 μM isoproterenol for 6 hours, and probed by immunoblot as indicated, with tubulin as a loading control. (D) Cell transplantations were done in the preadipose state as described in Experimental Procedures. After 6 weeks, fat pads were harvested and mRNA expression was analyzed by qPCR (n=5–8). For acute sympathetic stimulation, CL316,243, at 1mg kg−1, was injected intraperitoneally 5 hours before harvesting the transplanted fat pads. (E) Oxygen consumption in cultured brown, beige and white fat cells was assayed as described in Experimental Procedures. Oligomycin (ATP synthase inhibitor) was added to cells to measure the uncoupled respiration rate. The cells were treated with dibutyryl-cAMP for 12 hours before harvesting. Values are mean±SD (n=4). *, p<0.05; **, p<0.01; ***, p<0.001.