Figure 6.

Inhibition of GSK3 promotes activation of the MKK3/6-p38 MAPK-ATF2 signaling module. (a) Immunoblot analysis for activating phosphorylations of proteins of the MKK3/6-p38 MAPK-ATF2 signaling module in primary brown adipocytes pre-treated with 10 μM SB216763 for 1 h before stimulation with 0.1 μM ISO for 1 h. (b) Immunoblot analysis of total and phosphorylated p38 MAPK in immortalized brown adipocytes overexpressing GSK3β mutants or empty vector. Cells were treated with 0.1 μM ISO for 1 h. (c) Fgf21 expression in primary brown adipocytes pre-treated with 10 μM SB202190 (p38 MAPK inhibitor) for 1 h before treatment with 10 μM SB216763 (GSK3 inhibitor) for 1 h, followed by stimulation with 0.1 μM ISO for additional 6 h. (d) Schematic presentation of the proposed mechanism through which GSK3 regulates the thermogenic gene program in brown adipocytes. Data presented as mean of means +SEM (n = 3). 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. For panels a and b, full-length blots/gels are presented in Supplementary Fig. 2.