Penicillin and streptomycin

Human HCT116, SW480, and murine MC38 CRC cells were maintained at 37 °C in 5% CO₂ and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (VWR, Radnor, PA). HCT116 p53+/+ and p53−/− cells were a kind gift from Professor Bert Vogelstein at the Johns Hopkins University. For generating stable cell lines, MC38 cells were transfected with a mouse shTFRC plasmid (TRCN0000375695, Sigma, St. Louis, MO) or a mouse shPOLD1 plasmid (TRCN0000428858, Sigma) and selected with 1 µg mL⁻¹ puromycin.

Caco-2 cells (ATCC HTB-37) were grown in Dulbecco’s Modified Eagle’s Medium (VWR, Vienna, Austria) supplemented with 10% FCS, 2 mM glutamine (VWR, Vienna, Austria), 50 mg/l each penicillin and streptomycin (VWR, Vienna, Austria) and 1% non-essential amino-acids (VWR, Vienna, Austria). Cells were seeded onto 24-well Transwell™ membrane inserts (Corning, USA) placed in 24-well companion plates (Corning, USA), 24-well plates (for resazurin assay) or 3 cm Ø dishes (for glucosylation assay) and grown until day 21 after confluence to form a monolayer of polarized epithelial cells. In all incubation solutions containing clostridial toxins, the FCS concentration was lowered to 0.1%.

Isolated human monocytes were allowed to rest for 24 h prior to stimulation in RPMI 1640 Medium (Gibco, ThermoFisher, Paisley, UK) supplemented with 1% L-glutamine (Gibco), 1% penicillin/streptomycin (Gibco) and 5% de-complemented human serum.
THP-1 cell line (Sigma–Aldrich, St-Louis, MO, USA), a human monocytic cell line derived from monocytic leukemia patients, was cultured in RPMI 1640 supplemented with L-glutamine, 10% fetal calf serum (FCS; VWR International Ltd, Lutterworth, UK) and 1% penicillin and streptomycin (PenStrep) at 37 °C with 5% CO₂.
THP-1 or monocytes were seeded into 24-well plates (6 × 10⁵cells/mL) in RPMI 1640 and were left untreated or were treated with 20 ng/mL interleukin-4 (IL-4; Fischer Scientific, Loughborough, UK), 20 ng/mL interleukin-10 (IL-10; PeproTech, London, UK) or 100 ng/mL lipopolysaccharide (LPS; Sigma–Aldrich) for 24 h or in some experiments for the time of 48 h. Cells were washed following stimulation to ensure serum was removed.

We prepared and cultured mouse primary osteoblasts according to previously reported protocols (Bone research protocol p22-23). Primary osteoblasts were isolated from the femurs of two-month-old male mice. Briefly, femurs without muscle and connective tissue had their bone marrow thoroughly flushed out with PBS, using a 5-mL syringe and a 27-gauge needle, before cutting the clean diaphyses into little pieces of approximately 1–2 mm using scissors. The bone pieces were washed several times with PBS, and incubated three times in 0.1% collagenase (Life Technologies, Carlsbad, CA, USA) and 0.25% trypsin (Life Technologies, Carlsbad, CA, USA) at 37 °C in a shaking water bath in order to remove all remaining soft tissue and adherent cells, every 30 min. From the fourth time to the seventh time, the cells were collected and cultured with alpha Modified Eagle’s Medium (α-MEM) containing 10% fetal bovine serum (FBS, Corning, Steuben, NY, USA), and 1% penicillin and streptomycin (Amresco, Fried, WA, USA). Medium was changed three times per week, and, after approximately 7–10 days, cells reached subconfluency, at which point they could be used for experiments.