Fig. 3: Role of CREB transcriptional co-activators in the upregulation of FoxO1 expression by high-dose GluOC in 3T3-L1 adipocytes.

a Immunoblot analysis of cytoplasmic and nuclear fractions isolated from 3T3-L1 adipocytes after stimulation with GluOC (5 or 40 mg/ml) for 6 h. The blot is representative of five independent experiments. The relative amount of cytoplasmic and nuclear fractions analyzed was adjusted so as to obtain appropriate band intensities. Lamin B1 and α-tubulin were examined as loading controls for nuclear and cytoplasmic fractions, respectively. b HTRF analysis of the p300–CREB interaction in 3T3-L1 adipocytes incubated first in the absence or presence of 10 μM myristoylated PKI 14-22 amide or 10 µM U0126 and then in the additional absence or presence of GluOC (5 or 40 ng/ml) for 6 h. Data are means + SEM from 10 independent experiments. **p < 0.01 versus the value for control cells (vehicle); ^#p < 0.01 versus the value for GluOC (40 ng/ml) alone (two-way ANOVA followed by the Tukey–Kramer HSD test). c ChIP-qPCR analysis of the binding of CREB to the CRE-containing promoter region of the FoxO1 gene in 3T3-L1 adipocytes incubated with the indicated concentrations of GluOC for 6 h. Data are means + SEM from three independent experiments. *p < 0.05 versus the value for control (vehicle-treated) cells (one-way ANOVA followed by the Tukey–Kramer HSD test). d Immunoblot analysis of FoxO1 in 3T3-L1 adipocytes incubated with the indicated concentrations of garcinol for 24 h and then in the additional absence or presence of GluOC (5 or 40 ng/ml) for 6 h. A representative blot and quantitative data (means + SEM, normalized by the amount of β-actin) from three independent experiments are shown. **p < 0.01 versus the value for cells exposed to GluOC (40 ng/ml) alone (two-way ANOVA followed by the Tukey–Kramer HSD test)