Mccoy s 5a medium

The human colon carcinoma cell line, HT29 (ATCC, Manassas, VA, USA) were cultured in McCoy’s 5A medium (ATCC) containing 16.7 mmol/L glucose, 200 U/mL penicillin, 200 μg/mL streptomycin, and 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO₂. Cells were sub-cultured every 2 to 3 days. Before the start of experiments, HT29 cells were synchronized by incubation with 0.5% fetal calf serum (FCS) for 24 h.
All experiments were performed under proliferating conditions by incubating HT29 cells for 24 h at 37°C in the humidified atmosphere of 5% CO₂ in a sub-confluent state (75% confluence and with 10% FBS), in both the absence and presence of pectin (P7536, Sigma-Aldrich Co., St. Louis, MO, USA). This pectin was originally extracted with hot acidic water. Pectin was prepared by diluting 0.06 mg pectin powder in 1 mL McCoy’s 5A medium (309 μmol/L final concentration) and heating at 37°C for 30 min. HT29 cells were incubated with pectin for 24 h at 37°C.
At the end of the incubation period, HT29 cells were lysed in a buffer containing 20 mmol/L Tris-HCl, 137 mmol/L NaCl, 10% glycerol, 1% Nonidet-P40, 1 mmol/L phenylmethylsulfonyl fluoride, 10 mmol/L ethylenediaminetetraacetic acid, and 2 mmol/L sodium orthovanadate, and were immediately frozen at −80°C until molecular parameters were determined.

Human normal bone cell line hFOB and osteosarcoma cell lines MG-63 and U2OS were purchased from American Type Culture Collection (ATCC). According to the supplier's protocol, hFOB cells were cultivated in a mixture of 45% Dulbecco's modified Eagle's medium, 45% Ham's F12 Medium and 10% fetal bovine serum (all Sigma-Aldrich; Merck KGaA), U2OS cells were cultured using ATCC-formulated McCoy's 5A Medium (cat no. 30-2007; ATCC) containing 10% fetal bovine serum, whilst MG-63 cells were cultured with Eagle's Minimum Essential Medium (cat no. 30-2003; ATCC) containing 10% heat-inactivated fetal bovine serum (Sigma-Aldrich; Merck KGaA) maintained in a humidified atmosphere at 37°C under 5% CO₂. Cells were harvested upon reaching the logarithmic growth phase for subsequent experiments.

The female human colorectal adenocarcinoma intestinal epithelial cell (IEC) line HT-29 (ATCC, Manassas, VA, USA) was seed in T75 culture flask (Corning, Tewksbury, MA, USA) with McCoy's 5A medium (ATCC, Manassas, VA, USA) containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (P/S; original solution at 10,000 U/ml) (Gibco, Carlsbad, CA, USA). Cells were split every 4-6 days when reaching 90-95% confluence using Trypsin-EDTA (Gibco, Carlsbad, CA, USA). No cell authentication was performed on the HT-29 cell line.

Three human osteosarcoma cell lines, Saos-2 (HTB-85), MG-63 (CRL-1427), and U2OS (HTB-96) were procured from ATCC (Manassas, USA). Saos-2 and U2OS cells were cultivated in McCoy’s 5A Medium (30–2007, ATCC) and added with 10% FBS (Gibco, Waltham, USA). MG-63 cells were cultivated in Eagle’s Minimum Essential Medium (EMEM, 30–2003, ATCC) and added with 10% FBS (Gibco). Normal human osteoblast hFOB1.19 (CRL-11372) was procured from ATCC and cultivated in a 1:1 mixture of Ham’s F12 Medium Dulbecco’s Modified Eagle’s Medium (ATCC) containing 10% FBS (Gibco). All cell types were cultivated at 37°C in a humidified atmosphere containing 5% CO₂.