Anthocyanins inhibit cell invasion by regulating metastasis-related proteins in vivo and in vitro . (a) Hep3B cells were serum starved for 12 h and grown to confluence on a 6-well plate. Monolayers were wounded with a pipette tip, pretreated with IGF-1 (100 ng/ml) with or without Compound C for 30 min, and then treated with anthocyanins for 24 h. Images of wound closure were captured under a phase-contrast microscope after 24 h. (b) Hep3B cells were pretreated with IGF-1 (100 ng/ml) for 30 min, treated with anthocyanins for 24 h, and plated onto the apical side of Matrigel-coated filters in serum-free medium with or without IGF-1 and/or anthocyanins. Medium containing 2% FBS was placed in the basolateral chamber to act as a chemo-attractant. After 24 h, cells on the apical side were wiped off using a Q-tip, and cells that had migrated to the bottom of the filter were stained using crystal violet, and then photographed. (c) Cells were incubated with anthocyanins as described in (c) and Figure 4c, medium was collected, and MMP-2 activity was measured by zymography. (d) Mice were sacrificed, tumor extracts were prepared, and immunohistochemical analysis was used to quantify CD31 expression (e) Hep3B cells were transiently transfected with AMPK siRNA or non-specific siRNA for 72 h, pretreated with IGF-1 (100 ng/ml) for 30 min, treated with anthocyanins (200 μg/ml) for 24 h, and then subjected to Western blot analysis using antibodies against MMP-9, VEGF, Ang-1, AMPK and β-actin (loading control).