Trypsin

Adult NHDF were obtained from Coriell (Camden, NJ, USA). Dermal fibroblasts from patients with PXE were provided according to the authors’ description in [28 (link)], who also listed the clinical characteristics of the PXE patients. The study was approved by the Ethics Committee of the HDZ NRW, Department of Medicine, Ruhr University of Bochum (registry no. 32/2008, approval date is 3 November 2008). Primary cells were maintained under standardized conditions (37 °C, 5% CO₂) as a monolayer culture in tissue culture dishes (100 × 20 mm, Greiner bio-one, Frickenhausen, Germany) with Dulbecco’s modified Eagle’s medium without phenol-red addition (DMEM; Thermo Fisher Scientific, Waltham, MA, USA). DMEM was supplemented with either 10% (v/v) fetal calf serum (FCS; Biowest, Nuaillé, France) or 10% (v/v) lipoprotein-deficient FCS (LPDS) and 4 mM L-glutamine (PAN Biotech, Aidenbach, Germany), 1% (v/v) Penicillin-Streptomycin-Amphotericin B solution (100×; PAN Biotech, Aidenbach, Germany), as described previously [34 (link)]. The LPDS was prepared according to our previous work [28 (link)]. Medium changes were performed twice a week. The subculturing of near confluent primary NHDF was performed with an expansion ratio of 1:3 utilizing 0.05% (v/v) trypsin (PAN Biotech, Aidenbach, Germany) in Dulbecco’s phosphate buffered saline (PBS, 1×; Thermo Fisher Scientific, Waltham, MA, USA).

Human colon carcinoma cell lines HCT116 (CCL-247^TM), SW480 (CCL-228^TM) and human colon epithelium cell line FHC (CRL-1831^TM, used as control) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All the cell lines were maintained according to the distributor’s instructions, at 37 °C in humified atmosphere with 5% CO₂. Briefly, HCT116 cells were cultured in McCoy’s 5A (Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (FBS; Gibco) and gentamicin (50 µg/mL; Gibco). SW480 cells were cultured in Dulbecco’s Modified Eagle’s (DMEM; Sigma Aldrich, Saint Louis, MI, USA), supplemented with 10% FBS and 50 µg/mL of gentamicin. FHC cells were grown in DMEM:F12 (Gibco) supplemented with 10% FBS, 10 mM HEPES (Sigma Aldrich), 10 ng/mL cholera toxin (Sigma Aldrich), 0.005 mg/mL insulin (Sigma Aldrich), 0.005 mg/mL transferrin (Sigma Aldrich), 100 ng/mL hydrocortisone (Sigma Aldrich, USA) and gentamicin (50 µg/mL; Gibco). All the cells were passaged twice a week with 0.05% trypsin (PAN-Biotech GmbH, Aidenbach, Germany) in EDTA (Eurx, Gdansk, Poland) and regularly tested for Mycoplasma sp. contamination by PCR-ELISA test (Roche, Mannheim, Germany), according to manufacturer’s instruction.

NHDF from a 57- and a 55-year-old man, as well as a 50-year-old woman, were purchased from Coriell (Camden, NY, USA). Cells were cultivated as a monolayer culture on hard plastic cell culture dishes with complete medium consisting of Dulbecco’s modified Eagle´s medium supplemented with 10% (v/v) fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), 4 mM L-glutamine and 1% Penicillin-Streptomycin-Amphotericin B solution (100×; PAN Biotech, Aidenbach, Germany) under standardized conditions (37 °C, 5% CO₂). Medium changes were performed twice a week and fibroblasts were subcultured with 0.05% trypsin (PAN Biotech, Aidenbach, Germany) in Dulbecco’s phosphate buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA) after reaching 90% confluence.
Cells were subcultured in an expansion ratio of 1:3 on 100 × 20 mm plastic cell culture dishes and subsequently seeded at passage 7 in a cell density of 50 cells/mm². After 24 h, cells were incubated for 1 h with complete medium supplemented with 300 µM H₂O₂ (Carl Roth, Karlsruhe, Germany) or an appropriate volume of H₂O (control). Thereafter, cells were washed with PBS and further cultured in complete medium for an experiment-dependent time. For investigations on enzyme activity or protein level, cells were reseeded at a cell density of 50 cells/mm² and cultured for another 18 or 72 h in complete medium.