Figure 3.

GSK3 restricts the thermogenic program in brown adipocytes. (a) Expression of thermogenic genes (Fgf21, Ucp1, Dio2 and Ppargc1α) in primary brown adipocytes pre-treated with 10 μM SB216763 for 1 h before being stimulated with 0.1 μM ISO for an additional 6 h. (b) Immunoblot analysis of UCP1 in primary brown adipocytes pre-treated with 10 μM SB216763 for 1 h before being stimulated with 0.1 μM ISO for an additional 24 h. TFIIB serves as loading control. (c) Expression of thermogenic genes (Fgf21, Ucp1, Dio2 and Ppargc1α) in siRNA-transfected primary brown adipocytes stimulated with 0.1 μM ISO for 6 h. (d) Immunoblot analysis of GSK3α and GSK3β in siRNA-transfected primary brown adipocytes stimulated with 0.1 μM ISO for 24 h. TFIIB serves as loading control. (e) Representative normalized Seahorse run of oxygen consumption rates (OCR) in siRNA-transfected primary brown adipocytes. Quantification of basal (f) and ISO (1 μM)-induced (g) OCR. RT-qPCR data are presented as mean of means (+SEM) (n = 4). Statistical significance was determined by two-way ANOVA with repeated measures and Tukey’s post hoc test for multiple comparisons. *p < 0.05 versus H₂O. ^#p < 0.05 versus vehicle/scramble. Seahorse data are presented as mean (±SEM) of one representative experiment (e) or 3 independent experiments (f,g) and significance was determined by paired t-test (*p < 0.05). For panels b and d, full-length blots/gels are presented in Supplementary Fig. 2.