Hydrogen peroxide

HCT116 and LOVO cells were treated with MSC-CM or control medium for 24 h, and then fixed in 2% paraformaldehyde. The fixed cells or 5 μM-thick paraffin-embedded mouse colon sections were stained using antibodies against proliferation maker Ki67 (Bo Ao Seng, Beijing, China) or cell adhesion maker E-cadherin (Cell Signaling, Beverly, MA, USA). Briefly, the sections were dewaxed and rehydrated through a graded ethanol series, antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) for 15 min in a microwave, then the slides were incubated with 3% hydrogen peroxide for 10 min (Solarbio, Beijing, China), blocked in 5% BSA for 1 h, incubated with the primary antibodies overnight at 4 °C, followed by incubation with biotinylated secondary antibodies conjugated to streptavidin-horseradish peroxidase (ZhongShan Golden Bridge Biotechnology, Beijing, China). Immunoreactivity was visualized using DAB solution. For the tissue sections, the average number of Ki67 and E-cadherin positive cells was determined from five consecutive sections from the tumor region.

Samples collected from human patients and nude mouse model tumor tissue sections were de-paraffinized in xylene and rehydrated with gradient ethanol. After washing with double-distilled water, the tissue sections were incubated with 3% hydrogen peroxide (Solarbio, Beijing, China) for 10 min and antigenic repair was performed with sodium citrate antigen retrieval solution (Solarbio, Beijing, China). The sections were then blocked with 50% goat serum for 1 h and incubated with primary antibodies anti-E-cadherin (Proteintech, USA, 1:100), anti-Vimentin (Cell Signaling, USA, 1:200), anti-Gankyrin (Abcam, UK, 1:200) or anti-YAP1 (Proteintech, USA, 1:200) overnight at 4 °C before incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (Zhongshan Technology, Beijing, China) for 30 min at 37 °C. The tissue sections were stained with DAB (Zhongshan Technology) staining solution and counterstained with hematoxylin (LABEST, Beijing, China) for 10 s. Photographs were obtained using a Zeiss microscope (Carl Zeiss, Oberkochen, Germany).

The mouse xenograft tumors were fixed in paraformaldehyde for 1 h, followed by conventional paraffin‐embedding and sectioning (4 μm), and drying at 60℃. Paraffin sections were dewaxed with xylene and graded ethanol, and rinsed with distilled water. The slides were boiled in sodium citrate buffer (pH = 6.0, Solarbio, Beijing, China) for 10 min for antigen recovery. The sections were incubated in 3% hydrogen peroxide (Solarbio) for 10 min to block endogenous peroxidase activity and in 5% normal goat serum (ZGB‐BIO, Beijing, China) to block nonspecific protein binding sites. The sections were then incubated with antibodies against cleaved‐caspase‐3 (1:500; NB100‐56113, Novus Biologicals) and β‐Tubulin‐III (ab52623, RRID: AB_2819160, 1:800; Abcam) overnight at 4°C and subsequently incubated with goat antirabbit biotin‐conjugated secondary antibody (ab205718, 1:800; Abcam) for 0.5 h at room temperature. The reaction products were characterized using diaminobenzidine (Sigma‐Aldrich Chemical Company, St Louis, MO, USA). Nuclei were counted lightly with hematoxylin (Solarbio), and immunostaining was observed under a light microscopy (Olympus Optical Co., Ltd., Tokyo, Japan). The mean OD values of different sections were compared by Image‐pro Plus software.

STZ (Sigma, St. Louis, MO), hydrogen peroxide (Solarbio Science & Technology CO., Ltd, Beijing, China), LiCl (Royalton, Dalian, Liaoning Province, China ), xylene, hematoxylin, eosin Y, alcohol, ethanol, terminal deoxyribonucleotide transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay kit (In Situ Cell Death Detection Kit, Roche, Mannheim, Germany), Triton X-100 (Beyotime Institute of Biotechnology, Nantong, Jiangsu Province, China), Total RNA Extraction Kit (Tiangen Biotech Co., Ltd., Beijing, China), Super M-MLV Reverse Transcriptase and 2×Power Taq PCR MasterMix (Bioteke Corporation, Beijing, China), cDNA synthesis kit (IScript, BioRad, U.S.A.), RNase solid scavengers (Tiandz, Inc., Beijing, China), Powder agarose (D1 LE, Hispanagar, Spain), 50×TAE buffer (50 mM EDTA, 2 M Tris base, and 1 M glacial acetic acid).

Fucoidan standard extracted from U. pinnatifida (Fstd) was purchased from Sigma-Aldrich (Buchs, Switzerland). Sugar standards including _L-fucose, _D-galactose, and _D-glucose were purchased from Macklin (Shanghai, China). Synthetic antioxidants BHA, sodium hydroxide (≥99.0%), and ascorbic acid were also purchased from Macklin (Shanghai, China). Anhydrous ethanol (≥99.0%), hydrochloric acid (4 M), trifluoroacetic acid (≥99.0%), ferrous sulfate (9 mM), hydrogen peroxide (0.3%), ethanolic solution of salicylic acid (5 mM), potassium ferricyanide, 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-hydrazyl (DPPH) and dialysis bags were purchased from Solarbio (Beijing, China). All other chemicals used in this study were of analytical grade.