Fucoidan-Sargassum inhibits the initiation and assembly stages of invadopodia formation in HCCLM3 cells. (A) The elongated protrusions of the HCCLM3 cells, which are called invadopodia; the image was taken under an inverted microscope (400× magnification). (B) Western blotting analysis of FAK, Src, and Cortactin, which are key proteins involved in the initiation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h. (C) Confocal images of fixed control or Fucoidan-Sargassum-treated cells immunostained for actin (2nd panel), cortactin (middle panel), and Src (4th panel). The arrowheads indicate active invadopodia initiation in the control cells and the cytoskeletal changes in the treated cells. Scale bars: 7.5 µm. (D and E) Western blotting analysis of TKS5, N-WASP, ARP3, ARP2, and CDC42, which are key proteins involved in the assembly stage of invadopodia formation, and MMP2 and MT1-MMP, which are involved in the maturation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h.