Non essential amino acids (neaa)

The JIMT-1 human breast carcinoma cell line (ACC589) was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The normal-like breast epithelial MCF-10A cell line (CRL-10317), the cancer cell lines MCF-7 (HTB-22) and HCC1937 (CRL-2336) were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were tested negative for mycoplasma (Eurofins, Konstanz, Germany).
The JIMT-1 cells were routinely cultured at 37 °C in a humidified incubator with 5% CO₂ in air. The cells were cultured in DMEM/Ham’s F-12 medium supplemented with 10% fetal bovine serum (FBS), glutamine (2 mM), non-essential amino acids (1 mM), insulin (10 μg/ml), penicillin (100 U/ml), and streptomycin (100 μg/ml).
MCF-10A, MCF-7, and HCC1937 cell lines were cultured in RPMI 1640 medium (VWR) supplemented with 10% heat-inactivated FBS (VWR, Lund, Sweden), glutamine (2 mM), 1 mM non-essential amino acids (VWR), 10 μg/ml insulin (Sigma-Aldrich, Stockholm, Sweden), and 100 U/ml penicillin/100 μg/ml streptomycin (VWR). The MCF-10A cells were also supplemented with 20 ng/ml epidermal growth factor (Sigma-Aldrich), 50 ng/ml cholera toxin (Sigma-Aldrich), and 250 ng/ml hydrocortisol (Sigma-Aldrich). Finally, the HCC1937 medium was supplemented with 20 ng/ml epidermal growth factor (Sigma-Aldrich) besides the mentioned supplements.

Mouse SCCVII cells were transfected with 3 different vectors to express the HPV oncoproteins E6, E7 or both E6/E7 in the Radiation Oncology Department at the Université Catholique de Louvain (Prof. Vincent Grégoire), as previously described [26 (link)]. The RAW cell line is a murine macrophage cell line that originates from murine blood and is a kind gift from Prof. Alexandre Legrand (University of Mons). This cell line was cultured in DMEM high-glucose medium supplemented with 10% fetal calf serum (VWR International, Leuven, Belgium), 1% penicillin-streptomycin and 1% non-essential amino acids (ThermoFisher Scientific).

MOG-R3 peptide (MEVGWYRSPFSRVVLHLYRNGKRRR) was synthesized by Genscript at > 95%purity (Piscataway, NJ). CpG DNA (5’-T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*C*G*T*T*T*-3’) and GpG DNA (5’-T*G*A*C*T*G*T*G*A*A*G*G*T*T*A*G*A*G*A*T*G*A*-3’) was purchased from Integrated DNA Technology (Coralville, IA). β-mercaptoethanol, ethylenediaminetetraacetic acid (EDTA), 5x tris-borate-EDTA (TBE) buffer, and bovine serum albumin (BSA) were purchased from Sigma Aldrich (St. Louis, MO). Molecular biology grade water, fetal bovine serum (FBS), 20X phosphate buffered solution (PBS), 20% sodium dodecyl sulfate (SDS), HEPES, and non-essential amino acids were purchased from VWR (Radnor, PA). RPMI-1640 media, SYBR Gold gel stain, l-glutamine, penicillin-streptomycin, Ultrapure Agarose, Brilliant Violet 450 viability stain, and cell proliferation dye eFluor450 were purchased from Thermo Fisher Scientific (Grand Island, NY).

Rat aortic smooth muscle (A10) cells were purchased from ATCC and used as described (8 (link)). Wystar Kyoto rat 3M22 (WKO-3M22) cells, originally obtained from ATCC, were a generous gift from Dr Marsh Rolle. MEFs were a generous gift from Dr Liu Libin (Boston University School of Medicine). WKO-3M22 cell lines were cultured in high-glucose DMEM (Corning) without L-glutamine with 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% nonessential amino acids (VWR), and 1% L-glutamine (VWR). MEFs were cultured in high-glucose DMEM (Corning) without L-glutamine with 10% FBS, 1% sodium pyruvate, and 1% penicillin–streptomycin. All cells were incubated at 37 °C in 5% CO₂.

The JIMT-1 cell line [19 ] (ACC589) was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Upon receipt, the cells were thawed and amplified, and ampules were frozen in a liquid nitrogen container. The JIMT-1 cells were routinely cultured in DMEM/Ham’s F-12 medium (VWR, Lund, Sweden) supplemented with 10% fetal bovine serum (FBS) (VWR), 1 mM non-essential amino acids (VWR), 10 μg/mL insulin (Sigma-Aldrich, Stockholm, Sweden), 1 mM L-glutamine (VWR), and 100 U/mL penicillin/100 μg/mL streptomycin (VWR). The JIMT-1 cells were seeded at the density of 0.015 × 10⁶ cells/cm² in tissue culture vessel containing 0.2–0.3 mL per cm² of medium, and then kept at 37 °C in a humidified incubator with 5% CO₂ in air. After 4 days of culturing, the cell density was around 0.133 × 10⁶ cells/cm², i.e. the cells had gone through approximately 3 population doublings. The lag phase of the cells after trypsinization is 24 h, giving a population doubling time of around 24 h. The JIMT-1 cells were kept under strict surveillance for these proliferation characteristics and also morphology and only used for 50 passages and then a new ampoule was thawed. The cells were tested for mycoplasma and were found to be negative (Eurofins GATC Biotech, Konstanz, Germany).