Fig. 3. The protective effect of zingerone on Pi-induced calcification mediated by AMPK. VSMCs were treated with zingerone (100 nM) for 0.5, 1, 3, 6 and 12 hours (A). Cells were treated with or without Pi, zingerone, and Compound C (1 µM) for 2 days or 2 weeks (B, C) Protein levels of p-AMPK, AMPK, Dlx5, and CBFA1 were measured by Western blot analysis (A, B, left panel). Densitometry analysis was performed with the indicated antibodies (A, right panel and B, right panel, down panel). Alizarin red S staining (C). Data represent the mean±standard error of the mean of 3 individual experiments.

Pi, inorganic phosphate; AMPK, AMP-activated protein kinase; p-AMPK, phospho-AMPK; t-AMPK, total AMPK; VSMC, vascular smooth muscle cell; Dlx5, distal-less homeobox 5; CBFA1, core-binding factor α-1.

Statistical significance was determined relative to a control (^*p<0.05; ^†p<0.01; ^‡p<0.001) or compared with the Pi-treated group (^§p<0.05; ^∥p<0.01), Pi and zingerone group (^¶p<0.05; ^**p<0.01; ^††p<0.005) using the Student's t-test.