L glutamine

In this study, OvCa cell lines A2780 and SK-OV-3 (ATCC, Manassas, VA, USA) and human fetal lung fibroblasts cell line (MRC-5 pd19; ECACC, Salisbury, United Kingdom) were used to evaluate the antiproliferative activity of the tested compounds. OvCa cell lines were cultivated in RPMI 1640 containing 25 mM HEPES and 5 mM l-glutamine with 10% fetal bovine serum (FBS) (all provided from Biowest, Nuaillé, France) and 1% penicillin-streptomycin (Merck KGaA, Darmstadt, Germany). MRC-5 pd19 was cultured in DMEM supplemented with 10% FBS, 2 mM l-glutamine (all provided from Biowest, Nuaillé, France), 1% penicillin-streptomycin and 1% non-essential amino acids (NEAA) (both provided from Merck KGa, Darmstadt, Germany). To generate the cisplatin-resistant cell lines (A2780 CDDP; SK-OV-3 CDDP), increasing doses of cisplatin (CDDP) (Teva Pharmaceutical Industries Ltd. Petach Tikwa, Israel) were added to the culture medium, starting from the concentration of 100 ng mL⁻¹. Then, the cells were exposed to CDDP (3 cycles of 3 days each). After that, the cell culture medium was replaced with the fresh one without drugs for the next 3 days or one week until the cells recovered. After the 3 cycles, the dose of cisplatin was doubled until the concentration of 1000 ng·mL⁻¹ was achieved. Then, to maintain a resistant phenotype, 1000 ng·mL⁻¹ of CDDP was added once per 2 weeks for 3 days.

Hair Follicle Dermal Papilla Cells (HFDPC) (C12071, Promocell, Heidelberg, Germany) were cultured at 10,000 cells/cm² from passage 2 to passage 6 in 75 cm² T-flasks in Follicle Dermal Papilla Cell Growth Medium (C26501, Promocell), which contains fetal calf serum, bovine pituitary extract, bFGF and insulin, supplemented with L-Glutamine (X0550, Biowest, Nuaillé, France) and 1% (w/v) Penicillin/Streptomycin (P/S) (L0022, Biowest). Human Foreskin Fibroblasts (HFF) and HeLa cells were cultured at 10,000 cells/cm² for not more than 10 passages in DMEM (DMEM-HXA, Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% FBS (S1810; Biowest), L-Glutamine and P/S. Adipose-derived stem cells (ADSC) were cultured from passage 2 to passage 6 at 10,000 cells/cm² in ADSC Basal Medium (PT-3273, Lonza, Basel, Switzerland) with SingleQuots^TM Growth Supplement Kit (PT-4503, Lonza). Cultures were maintained at 37 °C and 5% CO₂ in a humidified atmosphere and passaged at 80% confluency.

The murine RAW264.7 (ATCC: TIB-71) macrophage
cell line was cultured in Dulbecco modified Eagle’s medium
(DMEM) supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 U/mL
streptomycin, 2 mM l-glutamine, and 1 mM sodium pyruvate
(all from Biowest). The human THP1 (ATCC TIB-202) monocyte cell line
was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium
supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 U/mL streptomycin,
2 mM l-glutamine, and 1 mM sodium pyruvate (all from Biowest).
Both these cell lines were maintained under standard culture conditions:
37 °C, 5% CO₂, and 90% relative humidity.

HaCaT cells [27 (link)], obtained from ATCC, were cultured in full medium (FM) comprising Dulbecco’s modified eagle high-glucose medium (Biowest, Nuaillé, France), completed with 10% FBS (Thermo Fisher Scientific, Waltham, MA USA), 1000 units/mL penicillin, 1000 μg/mL streptomycin (Sigma-Aldrich, St Louis, MO, USA) and 1% l-glutamine (Biowest, Nuaillé, France), at 37 °C in a 7.5% CO₂ controlled atmosphere. Where indicated, cells were changed to serum deprived medium (SS). TGF-β1 (PeproTech, Rocky Hill, NJ, USA) was inoculated at 2 ng/mL. For samples maintained continuously in the absence or presence of TGF-β, culture medium was refreshed every 24 h and new TGF-β1, if indicated, was inoculated. Epidermal Growth Factor (EGF; Sigma-Aldrich, St Louis, MO, USA) was supplemented at 10 ng/mL. Supplementation with FBS was achieved by introducing fresh FBS into SS samples up to a final 10% concentration, or 10% extra supplementation making up to 20% final when introduced to cells already in FM.

TC28A2 human chondrocyte cell line was maintained in high glucose Dulbecco’s Modyfied Eagle Medium (DMEM) with l-glutamine (Biowest, Nuaillé, France) and supplemented with 10% Fetal Bovine Serum (FBS) South America Heat Inactivated (Biowest, Nuaillé, France), 200 U/mL penicillin, and 200 µg/mL streptomycin. Mouse osteoblast cell line (7F2) was cultured in Minimum Essential Medium Eagle–alpha modification (α-MEM) without nucleosides (Biowest, Nuaillé, France). To obtain a full cultured medium, α-MEM was supplemented with 10% FBS and 2mM stable glutamine (Biowest, Nuaillé, France). TC28A2 and 7F2 cell lines were incubated in standard conditions at 37 °C in humidified atmosphere of 5% CO₂ and 95% air. Passive cells were used three times in the experiments.

The human non-small cell lung cancer cell line H358 was purchased from ATCC (ATCC-CRL-5807; ATCC, Manassas, VA, USA) and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS; c.c.pro, Oberdorla, Germany), 2 mM L-glutamine (Biowest, Nuaillé, France), 1% non-essential amino acids (NEAA, Biowest), and 1% penicillin-streptomycin (Biowest) and maintained at 37 °C in a humidified atmosphere with 5% CO₂. When confluent, cells were washed with phosphate-buffered saline (PBS, Biowest) and then harvested using trypsin-EDTA (Biowest).