Figure 4

Anthocyanins inhibit GSK3β and β-catenin in an AMPK-dependent manner. (a) Hep3B cells were serum-starved for 12 h, treated with IGF-1 (50–200 ng/ml) for 24 or 48 h, and then stained with trypan blue. Viable cells were counted under a hemacytometer. (b) Hep3B cells were serum-starved for 12 h, treated with IGF-1 (50–200 ng/ml) for 24 h, and then subjected to Western blot analysis using antibodies against p-GSK3β, β-catenin and β-actin (loading control). (c) Hep3B cells were serum-starved for 12 h, pretreated with IGF-1 (100 ng/ml) for 30 min, and treated with anthocyanins (50–100 μM) for 24 h. Total proteins were subjected to Western blot analysis using antibodies against p-GSK3β, GSK3β, p-AMPK, AMPK, β-catenin and β-actin (loading control). (d) Hep3B cells were serum-starved (for 12 h), pretreated with IGF-1 (100 ng/ml) and 10 μM of Compound C for 30 min, and then treated with anthocyanins for 24 h. Western blot analysis was used to compare protein levels. (e) Hep3B cells were serum-starved for 12 h, pretreated with IGF-1 (100 ng/ml) and 2 μM of BIO for 30 min, and treated with anthocyanins for 24 h. Western blot analysis was used to compare protein levels.