Bis acrylamide

Chondroitinase (CSase) ABC from Proteus vulgaris, hyaluronidase from sheep testes, heparinase (Hepase) I, Hepase II, Hepase III, and CSase B from Flavobacterium heparinum, protease from Streptomyces griseus, CS-A from bovine trachea, CS-C from shark cartilage, sodium cyanoborohydride, D₂O, Alcian Blue, NaH₂PO₄ and 2-Aminobenzamide (2-AB) were purchased from Sigma Aldrich (Shanghai, China). Keratan sulfate hydrolase (KSase) was prepared as in previous research [29 (link)]. Hep (200 IU/mg) from porcine intestinal mucosa were provided by Dongying Tiandong Pharmaceutical Co., Ltd (Dongying, China). Unsaturated disaccharides CS standards were purchased from Iduron (Manchester, UK). CS-D from shark fins was purchased from Seikagaku Corp. (Tokyo, Japan). Ethanol, acetone, NaCl, NaOH, trichloroacetic acid, ether, NH₄HCO₃, sodium acetate and acetic acid were purchased from China National Pharmaceutical Group Co., Ltd. (Shanghai, China). Acrylamide and bis-Acrylamide were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Superdex^TM Peptide 10/300 GL column was obtained from GE Healthcare Life Sciences (Beijing, China). YMC-Pack PA-G column (250 × 4.6 mm ID) was purchased from YMC (Kyoto, Japan). All other reagents were of the highest quality available.

Substrates with different stiffness were prepared as described previously by adjusting the ratio of 40% acrylamide to 2% bis-acrylamide (w/v) (Sangon Biotech, China) (Chen et al., 2019 (link); Tian et al., 2019 (link)). Tetramethylethylenediamine (TEMED, Klamar, China) and 10% ammonium persulfate solution (APS, Sinopharm, China) were used as cross-linking reagent. Before polymerization, 200 μl mixture was added onto a glass slide pre-treated with gel slick (Lonza, Switzerland), and a piece of silicified cover slip was put upon the gel solution to make a sandwich. After 5 min, the substrates were treated with sulfo-SANPAH (Thermo Fisher, USA) and coated with 1 mg/mL collagen I (Corning, USA) to facilitate the cell adhesion. Finally, the substrate was sterilized by 75% alcohol and UV before cell culture.

As shown in Supplementary Table 1, the concentrations of acrylamide and bis-acrylamide (79-06-1, 110-26-9, Sangon, Shanghai, China) were altered to establish polyacrylamide hydrogels of different stiffness as described previously [31 (link)]. In brief, the coverslips were incubated with 3-aminopropyltrimethoxysilane (APES, 919-30-2, Solarbio, Beijing, China), and glass slides were treated with dichlorodimethylsilane (DCDMS, 08471, Sigma-Aldrich, Burlington, Massachusetts). Acrylamide, bis-acrylamide and 0.1% TEMED (110-18-9, Sigma-Aldrich) were mixed with 1% ammonium persulfate (ST005, Beyotime) and then added onto glass slides and covered by coverslips before polymerization. Ten minutes later, the hydrogels were removed from the slides and placed into 6-well-plates for cell culture.