Dmem glutamax supplement

Primary Human Hepatocytes (HH, Lonza, Walkersville, MD, USA) were cultured in Hepatocyte Culture Medium (Lonza) according to the manufacturer’s protocol. HepG2 obtained from ATCC (Sigma-Aldrich, St Louis, MO, USA) were grown in CO₂ (5%) incubator at 37 °C in high glucose Dulbecco’s modified Eagle’s medium (DMEM) GlutaMAX Supplement (Thermo Fisher Scientific, San Jose, CA, USA) after addition of 10% (v/v) fetal bovine serum, 100 U penicillin, and 100 μg/mL streptomycin. Human cell lines were tested periodically for mycoplasma by using MycoAlert PLUS detection kit (Lonza). Cells were treated with 500 µM hydroxycitrate (HCA, Sigma-Aldrich) or 200 µg/mL red wine powder (RWP) obtained as previously described from the Italian red wine Aglianico del Vulture [21 (link)]. After 1 h of treatment with HCA or RWP, cells were stimulated with 5 ng/mL of TNFα (Sigma-Aldrich) for up to 24 h. Where indicated, cells were treated also with 20 μM IKK inhibitor VII (IKK 16, Sigma-Aldrich), 5 mM sodium acetate (Ac, Sigma-Aldrich) or 5 mM sodium malate (M, Sigma-Aldrich) alone or in combination with 500 μM NADPH (Sigma-Aldrich).

MN9D-Nurr1^Tet-on (iMN9D) cell line stably transfected with pBUD-IRES-eGFP (CTRL cells) or with pBUD-β-globin-MYC IRES-eGFP, 2xFLAG-α-globin (Hb cells) were used [10 (link)]. Cells were maintained in culture at 37 °C in a humidified CO₂ incubator with DMEM/F12 medium (Gibco by Life Technologies, DMEM GlutaMAX® Supplement Cat. No. 31966-021; F-12 Nutrient Mix GlutaMAX® Supplement Cat. No. 31765-027) supplemented with 10% foetal bovine serum (Euroclone, Cat. No. ECS0180L), 100 μg/ml penicillin (Sigma–Aldrich), 100 μg/mL streptomycin (Sigma-Aldrich). 300 μg/ml neomycin (Gibco by Life Technologies, Cat. No 11811-031) and 150 μg/mL Zeocin (Invivogen, Cat. No. ant-zn-05) were used for selection.

Human A549 cells (a kind gift from Dr J. McCauley, MRC-NIMR) were maintained in Dulbecco’s modified Eagle’s medium-high glucose, (DMEM GlutaMAX Supplement) (gibco-Life technologies) supplemented with 5% fetal calf serum (FCS). A549 cells were seeded on to 24-well plates and when confluent, used for experiments.

Cells were grown at 37 °C and 5% ambient CO₂. HeLa Kyoto (ATCC), MDA-MB-231 (ATCC), and A549 cells (ATCC) were grown to ~80% confluence in 140 × 20 mm dishes (Nunclon^TM Delta Airvent, Thermo Fisher) with Dulbecco’s Modified Eagle’s Medium, high glucose, GlutaMAX^TM supplement (DMEM, Thermo Scientific), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin. H-358 (ATCC CRL-5807) and U-2932 (ATCC) cell lines were cultured in RPMI 1640 with GlutaMAX^TM, 1% penicillin-streptomycin, and 10% FBS. Prior to HATRIC-LRC experiments with folate, HeLa cells were starved for 48 h in folate-free RPMI (Gibco).

Cells were grown at 37 °C and 5% ambient CO₂ to approximately 80% confluence in 140 × 20 mm dishes (Nunclon^TM Delta Airvent, Thermo Fisher, Waltham, MA, USA) with Dulbecco’s Modified Eagle’s Medium, high glucose, GlutaMAX^TM supplement (DMEM, Thermo Scientific, Waltham, MA, USA), 10% fetal bovine serum (FBS, BioConcept, Allschil, Switzerland), and 1% penicillin–streptomycin. For SILAC labeling, cells were grown in SILAC DMEM high glucose with stable glutamine and without arginine and lysine (Pan Biotech, Aidenbach, Germany), supplemented with 15% dialyzed FBS (Pan Biotech, Aidenbach, Germany), 1% penicillin–streptomycin, 10 mM HEPES (Gibco), non-essential amino acids (Gibco), 200 mg/L L-proline, 42 mg/L L-arginine or L-arginine [10 (link)], and 73 mg/L L-lysine or L-lysine [8 (link)] (Silantes, München, Germany) for 7 days, and the incorporation rate of heavy amino acids was determined to be 95–98% by MS analysis. For polarized cultures, 5 × 10⁶ or 0.5 × 10⁶ cells, respectively, were seeded into 75 mm or 12 mm polycarbonate Transwells^TM with a pore size of 0.4 µm (Corning) and cultured for 6–7 days while changing the medium every second day.