Foetal bovine serum (fbs)

All mouse ESC lines used in this study were grown at 37 °C and 5% CO2 on gelatinised plates. Dulbecco’s Modified Eagle Medium (Gibco) was supplemented with 15% foetal bovine serum (Labtech), 2 mM L-glutamine (Life Technologies), 1x penicillin-streptomycin solution (Life Technologies), 1x non-essential amino acids (Life Technologies), 0.5 mM beta-mercaptoethanol (Life Technologies), and 10 ng/mL leukaemia inhibitory factor. Cells were passaged using trypsin-EDTA (0.25%, Gibco) with 2% chicken serum. Cells were regularly tested for the presence of mycoplasma.
To induce conditional knockout, PRC1^CKO cells were treated with 800 nM 4-hydroxytamoxifen (OHT) (Sigma) for 72 h. To induce and maintain degradation and depletion of SUZ12, dTAG-SUZ12 were treated with 100 nM dTAG-13^{101 (link)} for 96 h.

All cancer cell lines used in this study were obtained from American Type Culture Collection (ATCC) and were not authenticated upon arrival. Mycoplasma testing was performed. Human foreskin fibroblasts (HFFF2) were obtained from Culture Collections (Public Health England, UK). All cell lines were grown in high glucose Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) except for HCT-116, SK-OV-3, Capan2 and HT29 that were grown in McCoys 5a medium, SNU-387, NCI-H23, OVCAR-3 that were grown in RPMI and A549, PC-3 that were grown in F12K medium. All media was supplemented with 10% foetal bovine serum (FBS) (Thermo Fisher) and 1% of antibiotic-anti-mycotic (Thermo Fisher). Human umbilical vein endothelial cells (HUVEC) from pooled donors were purchased from Promocell (Heidelberg, Germany). The cells were grown in M199 (Life Technologies, Grand Island, NY) with 20% foetal bovine serum (Labtech, Heathfield, UK), 10U/ml heparin (Sigma, St. Louis, MO), and 30 μg/ml endothelial cell growth supplement (Sigma). Experiments were performed using HUVECs between passage 3 and 5.

MG63 osteosarcoma cells (passage 17 to 28) were maintained in α-MEM Eagle with sodium bicarbonate, 10% Foetal Bovine Serum (LabTech, Heathfield, UK), 2 mM l-Glutamine and 100 mg/mL Penicillin–Streptomycin at 37 °C and 5% CO₂. At 90% confluence, cells were trypsinised, collected and seeded at 10,000 cells/cm² on ibiTreat µ-Slide 8 well, 24-well plates, 6-well plates, T12.5 or T25 with working volumes of 250 µL, 500 µL, 2.5 mL, 2.5 mL and 5 mL, respectively. To assess cell attachment, cells were seeded in the presence of different fucoidans as described below, and after 24 h, assessments were performed. To assess proliferation or cell viability, treatments with different fucoidans were applied 24 h after cell seeding.

iNOS supporting activity was measured using a novel mouse macrophage bioassay recently reported by us¹⁴ . Briefly, RAW 264.7 mouse macrophages (ATCC, USA), were cultured using Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich, UK) supplemented with 2mM L-glutamine (Sigma Aldrich, UK), nonessential amino acids (Invitrogen, UK) and penicillin-streptomycin (Sigma Aldrich, UK) at 5% CO₂ and 37 °C. At confluence, cells were scraped and spun at 400 relative centrifugal force for 5 minutes. 10% filtered foetal bovine serum (LabTech, UK) was included only when culturing. For the iNOS supporting bioassay, media was replaced with neat (100%) human plasma from patients before and at different times after cardiopulmonary bypass – this was added directly to RAW 264.7 cells. Cells were then stimulated to express iNOS with LPS (1 μg/ml) for 24 h. Plasma was then removed and immediately assays nitrite levels as a marker of iNOS activity. Nitrite was measured using the Griess assay as we have described previously¹⁴ .

Murine pre-osteoblast cells MC3T3-E1 (Riken cell bank, passage 22–23, [31 (link)]) were cultured in Minimum Essential Alpha Eagle medium (Lonza) supplemented with nucleosides and 2 mM UltraGlutamine I, 10% v/v foetal bovine serum (Labtech) and a solution of 100 units/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich). Cells were kept at 37°C and passaged when 70% confluent. Prior to AFM analysis, cells were seeded on tissue culture plastic dishes (D = 36 mm, seeding density 1200 cell/cm²) and tested after 4 to 5 days of culture when reaching 45%-85% confluence. If not confluent, MC3T3 cells are not expected to produce mineral and were, therefore, considered to be in the pre-osteoblastic differentiation stage [31 (link)]. MC3T3 cells are known to have an HA-rich glycocalyx involved in mechanotransduction [28 (link)] and to express CD44 [29 (link),30 (link)] under similar culture conditions. To the best of our knowledge, no information is available on the predominant CD44 isoforms or on the expression of the HA synthases in these specific cells, which is, however, expected to be similar to other cell types displaying an HA-rich glycocalyx [5 (link)–7 (link)].

RAW 264.7 mouse macrophages (ATCC, USA), were cultured using Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich, UK) supplemented with 2mM L-glutamine (Sigma Aldrich, UK), nonessential amino acids (Invitrogen, UK) and penicillin-streptomycin (Sigma Aldrich, UK) at 5% CO₂ and 37°C. At confluence, cells were scraped and spun at 400 relative centrifugal force for 5 minutes. 10% filtered foetal bovine serum (LabTech, UK) was included only when culturing.

SCs primary cultures were obtained from sciatic nerves dissected from 2-day-old Wistar pups, according to the protocol modified by Davis and Stroobant^{62 (link)}. In brief, sciatic nerves were digested with trypsin/collagenase (Type I, Sigma-Aldrich, St. Louis, MA Louis, MA) and seeded into T25 flasks with fresh high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, Poole, UK) containing 10% foetal bovine serum (LabTech, Uckfield, UK). To selectively remove fibroblasts, cells were treated with 1 mM cytosine arabinoside (AraC, Sigma-Aldrich, UK) for 48 h and then with anti-Thy 1.1 (1:1000, Serotec, Bio-Radgroup, Hercules, CA) and rabbit complement (1:2 v/v) (Cedarlane, ON, Canada). SCs were then amplified in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, Poole, UK), 10% FBS (LabTech, Uckfield, UK), 10 μM forskolin (Fsk; Sigma-Aldrich, UK) and 63 ng/ml glial growth factor-2 (GGF-2, Acorda, Ardsley, NY, USA).
SCs were incubated in 5% CO₂ at 37 °C and maintained at sub-confluent levels onto Poly-D-Lysine (PDL, Sigma-Aldrich, UK)-coated 75 cm² flasks.