Figure 4

Fucoidan induces mitochondrial membrane depolarization, increases the release of cytochrome c and Smac/Diablo from the mitochondria, and alters the levels of the Bcl-2 family proteins in HT-29 cells. (A) Cells were treated with various concentrations of fucoidan for 48 h as described in Figure 1, then subjected to subcellular fractionation. The resultant cytosolic fractions were analyzed via Western blotting with the indicated antibodies. Photographs of chemiluminescent detection of the blots, which were representative of three independent experiments, are shown. The relative abundance of each band to its own α-tubulin was quantified, and the control levels were set to 1. The adjusted mean ± SEM (n = 3) of each band is shown above each blot. (B) Cells were treated with fucoidan for 48 h. Cells were loaded with JC-1 and then analyzed via flow cytometry. The numbers of cells with normal polarized mitochondrial membranes (red) and with depolarized mitochondrial membranes (green) are expressed as a percentage of the total cell number. Each bar represents the mean ± SEM (n = 6). (C) Cells were treated for 36 h with various concentrations of fucoidan as described in Figure 1. Cell lysates were analyzed via Western blotting with the indicated antibodies. Photographs of chemiluminescent detection of the blots, which were representative of three independent experiments, are provided. The relative abundance of each band to its own β-actin was quantified, and the control levels were set to 1. The adjusted means ± SEM (n = 3) of each band are shown above each blot. (A, B, C) Means without a common letter differ, P < 0.05.