FIGURE 2.

AICAR increases β-catenin expression and phosphorylates GSK3β to stabilize β-catenin. A and B, β-catenin expression was determined in 3T3-L1 preadipocytes without or with AICAR (0. 5 mm) treatment for 48 h together without or with AMPK knockdown (KD) by RT-PCR and Western blotting. C, Western blot analysis of nuclear β-catenin levels in 3T3-L1 preadipocytes without or with AICAR (0.5 mm) treatment for 48 h. D, immunofluorescence staining analysis of 3T3-L1 preadipocytes with the anti-β-catenin antibody without (Ctrl, control) or with AICAR (0.5 mm) treatment for 24 h. Scale bars, 50 μm. E, the phosphorylation (Ser⁹) of GSK3β was determined without or with AICAR (0.5 mm) treatment at the indicated times together with or without AMPK knockdown. F, Oil Red O staining of 3T3-L1 adipocytes and quantification of lipid accumulation without or with AICAR (0.5 mm) treatment for 9 days together with or without β-catenin knockdown. Scale bars, 50 μm. G and H, GATA3 mRNA (G) and protein (H) levels were determined in 3T3-L1 preadipocytes without or with AICAR (0.5 mm) treatment for 48 h together without or with DKK1 treatment. I, ChIP assay of GATA3 binding at the PPARγ2 promoter (pro) under the specified conditions for 48 h. A neighbor region of the PPARγ2 gene without transcripts (Non-pro) was used as a negative binding control region, and nonspecific IgG was used as a negative control for chromatin pulldown. One-way ANOVA was used in A, F, G, and I, and other statistics were performed using Student's t test; bar graphs are mean ± S.E. In vitro data are the means of three independent experiments. *, p < 0.05; **, p < 0.01.