*4.5. Cell Migration Assay*

For cell migration assay, HT-29 cells were grown to 80% confluence, harvested, and seeded in 24-well Transwell plates with 8 μm pores at the density of 5 × 10<sup>4</sup> cells/well. DMEM containing 10% FBS was added to the lower chamber. After 24 h, 0, 200, 400, and 800 μg/mL of fucoidan was added to the upper chamber, and the cells were cultured for 24 h and 48 h, respectively. At the end of incubation, cells were stained with crystal violet, photographed, and counted under an inverted microscope (DM100FL, Leica, Germany).

#### *4.6. Quantification of Apoptosis with the Annexin V*/*Propidium Iodide Assay*

Cells in log-phase growth were seeded in six-well plates at a density of 1 × 10<sup>5</sup> cells/well. After 24 h, 0, 200, 400, and 800 μg/mL of fucoidan was added and the cultures were incubated for 48 h. The cells were then harvested, washed, and resuspended in the binding bu ffer containing Annexin V and propidium iodide (PI). After incubation for 15 min at room temperature, the stained cells were analyzed by flow cytometry. Data were analyzed using FlowJo software (10.0.7., BD, Franklin, NJ, USA).

#### *4.7. Total RNA Extraction and Reverse Transcription-Polymerase Chain Reaction*

RNA was isolated and reverse-transcribed into cDNA using the revert aid first strand cDNA synthesis kit, according to the manufacturer's protocol. For PCR, the cDNA was mixed with forward and reverse primers (for the list of primers, see Table 1) and dream taq green PCR master mix (2x). The amplification included 30 cycles of denaturation for 30 s at 95 ◦C, annealing for 30 s at 57 ◦C, and elongation for 30 s at 72 ◦C. The PCR products were subjected to electrophoresis on 1% agarose gel. The gel was analyzed using the Image J software, and the amount of cDNA was determined and normalized to the amount of β-actin cDNA.


**Table 1.** Primer sequence table.

#### *4.8. Silencing the Expression of DR4 by siRNA*

The cells in the logarithmic phase of growth were seeded in a six-well plate at a density of 2 × 105 cells/well. After 24 h, a mixture of 6 μL Lipo 8000 and 150 μL siRNA (sense: 5-GCU GUUCUUUGACAAGUUUTT3, antisense: 5 AAACUUGUCAAAGAACAGCTT-3) was added, and the medium was replenished to 2 mL. The mixture was aspirated after 6 h, and the cells were incubated with 0, 200, 400, and 800 μg/mL of fucoidan for an additional 48 h.

#### *4.9. Detection of Mitochondrial Membrane Potential by JC-1 Staining*

The cells in the logarithmic phase of growth were seeded in a six-well plate at a density of 6 × 10<sup>3</sup> cells/well. After 24 h, 0, 200, 400, and 800 μg/mL of fucoidan were added. The cells were stained 48 h later using the mitochondrial membrane potential detecting kit (JC-1); the manufacturer's protocol was employed. Stained cells were photographed and counted using a multi-function microplate reader.

## *4.10. Inhibition of Cytochrome C Expression*

The cells in the logarithmic phase of growth were seeded in a six-well plate at a density of 6 × 10<sup>3</sup> cells/well. Then, 3 μM cytochrome C inhibitor was added after 24 h. One hour later, the inhibitor was washed out, and the cells were incubated for an additional 48 h in the presence of 0, 200, 400, and 800 μg/mL of fucoidan.
