Fig. 4

ADM induced UCP1 expression. a, ADM induced UCP1 in a dose dependent manner. Protein expression was measured in hMADS-adipocytes grown in differentiation medium for 17 days. They were stimulated by increasing doses of ADM for 24 h. Expressions of UCP1 (upper panel) and Tubulin-βI (lower panel) used as a loading control were analyzed by Western blot using specific antibodies. Representative Western blots are shown. Full-length blots are presented in Supplementary Figure 9. b, Quantification of the signals. Protein expression was quantified using Quantity One Program and compared to the expression of Tubulin-βI. The means ± SEM were calculated from four independent experiments (*p < 0.05). c-e, Transcriptional activation of UCP1 promoter in response ADM. GFP expression was driven by the human UCP1 promoter in hMADS cells differentiated for 14 days. GFP fluorescence was determined after 4 more days of incubation with the indicated concentration of ADM. Control condition corresponds to cells that did not receive ADM. All images were recorded with the very same parameters. (Scale bar: 20 μM). f, Quantification of the signals. Means were calculated from 3 independent experiments performed in duplicate on at least 6 distinct recordings for each coverslip. The fluorescence signal measured as “raw integrated density” was divided by the number of nuclei present in the microscopic field. Rosiglitazone (BRL) was used as a positive inducer of browning. (*p < 0.05, **p < 0.01)