L glutamine

Human colon epithelial cells, Caco-2 (accession N^o 86010202, European collection of authenticated cell cultures (ECACC), Salisbury, UK), were grown in monolayers in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% inactivated calcium-free fetal calf serum (Gibco Laboratories, Grand Island, NY, USA), 100 U/mL penicillin/0.1 mg/mL streptomycin (PanBiotech GmbH, Aidenbach, Germany) and 2 mM L- glutamine (PanBiotech GmbH, Germany), at 37 °C and in an atmosphere containing 5% CO₂ (CO₂ Incubator; Thermo Scientific, Marietta, OH, USA) [28 (link)]. HaCaT (immortalized human keratinocyte) cells (accession No. 300493, Cell Lines Service GmbH, Eppelheim, Germany) were cultured in DMEM medium that was low in calcium (0.03 mM) and supplemented with 10% calcium-free fetal calf serum, 100 U/mL penicillin/0.1 mg/mL streptomycin and 2 mM L-glutamine (PanBiotech GmbH, Germany).

KBM-7 cells were cultured in IMDM supplemented with 10% fetal calf serum FCS (PAN-Biotech), 100 U/ml penicillin, 100 μg/ml streptomycin (PAN-Biotech) and L-glutamine (PAN-Biotech). HEK293T cells (DSMZ, Germany) were grown in DMEM high glucose supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 μg/ml), and L-glutamine (PAN-Biotech).

Human colonic epithelial cells, Caco-2 (accession No. 86010202, lot 16H030, ECACC), human Caucasian colon adenocarcinoma grade II cell line HT29-MTX-E12 (accession N° 12040401, lot 16D017, ECACC) and human Caucasian hepatocyte carcinoma cells HepG2 (accession No. 85011430, lot 16K046, ECACC) were cultured in monolayers in Dulbecco’s Modified Eagle Medium (DMEM) substituted with 10% heat-inactivated fetal bovine serum (FBS) (Gibco Laboratories, Grand Island, NY, USA), 100 U/mL penicillin/0.1 mg/mL streptomycin (PanBiotech GmbH, Hamburg, Germany) and 2 mM L-glutamine (PanBiotech GmbH, Germany). Cells were grown at 37 °C and 5% CO₂ (CO₂ Incubator, Thermo Scientific, Marietta, GA, USA) and sub-cultured when the monolayers reached 90% confluence. For sub-culturing, the monolayers were treated with trypsin–EDTA (PanBiotech GmbH, Germany).
The human Caucasian histiocytic lymphoma cell line U937 (accession No. 85011440, lot No. 11D008) was cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with heat-inactivated FBS (10%) penicillin (100 U/mL)/streptomycin (0.1 mg/mL) and L-glutamine (2 mM). Cells were grown at 37 °C and 5% CO₂ and passaged every 2–3 days.

BT-474 (ACC 64) and MCF-7 (ACC 115) breast cancer cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). MCF-10A (CRL-10317), a non-tumorigenic mammary epithelial cell line, was obtained from the American Type Culture Collection (ATCC). ZR-75-1 (CRL1500, ATCC) and MDA-MB-453 (ACC 65, DSMZ) cells were purchased from DSMZ. The identity of all cell lines was confirmed by DNA finger printing analysis utilizing the GenePrint 10 System (Promega). BT-474 and MDA-MB-453 cells were cultivated in DMEM medium (Pan-Biotech) supplemented with 10% FCS (Sigma-Aldrich), 2 mM L-Glutamine (Pan-Biotech) and 1% Penicillin/Streptomycin (Pan-Biotech). ZR-75-1 and MCF-7 cells were propagated in RPMI 1640 medium (Pan-Biotech) supplemented with 10% FCS, 2 mM L-Glutamine and 1% Penicillin/Streptomycin. In addition to other components, the medium for MCF-7 cells contained 1 mM Sodium-Pyruvate (Sigma-Aldrich). MCF-10A cells were cultured in DMEM/F12 medium (Pan-Biotech) supplemented with 5% Horse serum (Sigma-Aldrich), 1% Penicillin/Streptomycin, 20 ng/ml EGF (Sigma-Aldrich), 0.5 μg/ml Hydrocortisone (Sigma-Aldrich), 10 μg/ml Insulin (Sigma-Aldrich) and 0.1 μg/ml Cholera toxin (Sigma-Aldrich).

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).

Cells were cultured in DMEM/F12 (1:1) supplemented with 10% FCS and 2 mM L-glutamine (all cell culture reagents were from PAN-Biotech, Aidenbach, Germany, unless stated otherwise) and grown in a humidified 37 °C incubator with 5% CO₂. Medium was changed every 2–4 days. Cells were harvested by washing with PBS and detaching with trypsin when reaching about 70% confluence. To minimize alterations in morphology, proliferation, and response to stimuli, cells were maximally used up to passage number 45 and all experiments were performed within a range of 20 passages maximum.