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. 2009 Dec 22;285(10):7153–7164. doi: 10.1074/jbc.M109.053942

FIGURE 6.

FIGURE 6.

Only a subset of cells in rosiglitazone-treated white adipocyte cultures is UCP1-positive and demonstrates NE-induced UCP1-dependent thermogenesis. Primary cultures of white and brown adipocytes were grown for 7 (A–D) or 6 (E and F) days in the absence or presence of 1 μm rosiglitazone. A and B, cells were analyzed by immunofluorescence cell staining using antibodies against VDAC (red) and UCP1 (green). Nuclei were counterstained with Hoechst 33258 (blue). Cells were examined with a fluorescence microscope, and representative images are presented (lower (A) and higher (B) magnification). (A version of A and B, with an altered background, is found in supplemental Fig. 5.) C and D, protein levels of VDAC and UCP1 in control and rosiglitazone-treated white adipocyte cultures. C, representative Western blots. Total cell lysates (30 μg of protein/lane) were examined using antibodies against VDAC and UCP1. D, VDAC and UCP1 protein levels in control and rosiglitazone-treated white adipocyte cultures. The values represent means ± S.E. of four independent experiments, each performed in duplicate. The rosiglitazone-induced level was in each experiment set to 100%, and the levels in untreated cells were expressed relative to this value in each individual experiment. The significance of rosiglitazone treatment was determined with paired t test. **, p < 0.01; ***, p < 0.001. E, representative traces showing the effect of NE on oxygen consumption of control (thin line) and rosiglitazone-treated (thick line) white adipocyte cultures. At the arrow, 1 μm NE was added. F, NE-induced component of respiration (calculated as the difference between the rates of oxygen consumption before and after addition of NE) in control and rosiglitazone-treated white adipocyte cultures. The polarographic output was time-differentiated, sampled, and recalculated per milligram of total cellular protein. The values are means ± S.E. of four independent experiments. The significance of rosiglitazone treatment was determined with paired t test. **, p < 0.01.