Dulbecco s modified eagle s medium dmem

Both lipoma-derived stem cells (LDSCs) and adipose-derived stem cells (ADSCs) were isolated by enzymatic digestion of tissue samples, respectively, as we previously described [26 (link)]. Stromal vascular fraction (SVF) of cells, obtained from tissue homogenates after collagenase I digestion, was seeded in 25 cm² cell culture flask (Greiner Bio One, Kremsmünster, Austria) in standard cell culture medium that contained Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 2 mM stable glutamine and 1% antibiotic-antimycotic solution (all purchased from Capricorn Scientific, Ebsdorfergrund, Germany). Media were changed 16–18 h after isolation to remove non-attached cells. After reaching confluency, the first cell passage was performed (P1), which enabled purification of mesenchymal stem cells. Cells were cultured in standard cell culture conditions, meaning temperature of 37 °C and humidified atmosphere with the presence of 5% CO₂. Medium was changed every three days. Conditioned media (CM) of LDSCs (LDSC-CM) and ADSCs (ADSC-CM) were collected as a three-day medium just before passage 2 (P2) and stored at –80 °C until further analyses.

Folin-Ciocalteu’s phenol reagent, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, luminol, ethylenediaminetetraacetic acid (EDTA), H₂O₂, 3-(4,5-dimethythiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT), FeCl_3·6H₂O, and L-ascorbic acid were purchased from Sigma-Aldrich (St. Louis, USA). Various solvents such as ethanol (EtOH) and ethyl acetate (EtOAc) were of analytical grade. In addition, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, penicillin-streptomycin, and trypsin used for cell culture were purchased from Capricorn Scientific (Ebsdorfergrund, Germany).

Human non-small cell lung cancer (NSCLC) cell lines (H441, A549, H460, H727, H520, H332m) were kindly provided by Prof. Julian Downward and the small cell lung cancer cell line (SCLC) H82 was kindly provided by Prof. Roman Thomas. They were cultured in a humidified 37 °C atmosphere containing 10% CO₂ in RPMI 1640 medium (Thermo Fisher, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, Taufkirchen, Germany) and 1000 U/mL of both penicillin and streptomycin (Sigma Aldrich, Taufkirchen, Germany), and were tested for mycoplasma at regular intervals (mycoplasma barcodes, Eurofins Genomics, Ebersberg, Germany). HT22 cells (kindly provided by David Schubert, Cellular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Capricorn Scientific GmbH, Ebsdorfergrund, Germany) supplemented with 10% heat-inactivated FBS (Merck KGaA, Darmstadt, Germany), 100 U/mL penicillin, 100 mg/mL streptomycin (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and 2 mM L-glutamine (Merck KGaA, Darmstadt, Germany).

Coelenterazine h was obtained from NanoLight, Pinetop, AZ. Dulbecco’s Modified Eagle’s Medium (DMEM), PBS, penicillin/streptomycin, and Trypsin-EDTA were from Capricorn Scientific Gmbh, Ebsdorfergrund, Germany. Poly-L-lysine hydrobromide, FCS, L-glutamine, PEI, acetlycholine iodide, methyl-3-(dimethylamino)propionate (Mda), and trimethyl-(5-methyl-furan-2-ylmethyl)-ammonium iodide (Fur) were obtained from Sigma-Aldrich, Merck KGaA, Darmstadt, Germany. Arecoline hydrobromide, pilocarpine hydrochloride, and methacholine chloride were from TCI Chemicals, Eschborn, Germany, and guvacoline hydrobromide from TRC Canada, Toronto, Canada. Pertussis toxin was purchased from EMD Millipore Corp., Merck KGaA. FR900359 was isolated from Ardisia crenata leaves as previously described in Schrage et al., 2015 (link).

The mouse mammary epithelial HC11 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and the mouse embryonic fibroblast NIH3T3 cell line was kindly provided by Dr. Young Bong Kim (Konkuk University, Seoul, Korea). HC11 cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI 1640; Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS; Merck, Kenilworth, NJ, USA) and 1% penicillin/streptomycin (Capricorn Scientific). NIH3T3 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Capricorn Scientific) supplemented with 10% FBS and 1% penicillin/streptomycin. Both cell lines were grown in a humidified incubator at 37 °C under 5% CO₂ conditions.

The recombinant wild type PTEN and Trx1 were purified as previously described [27 (link)]. Thioredoxin reductase was purified from the mouse liver as described previously [49 (link)]. β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH), N-ethylmaleimide (NEM), N,N-dimethylformamide (DMF), and dl-dithiothreitol (dl-DTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrogen peroxide (H₂O₂) was purchased from OCI Company Ltd. (Seoul, Korea). tert-Butyl hydroperoxide (t-BHP) was purchased from Alfa Aesar (Ward Hill, MA, USA). Protease inhibitor (Complete ULTRA Tablets) was purchased from Roche Diagnostics GmbH (Indianapolis, IN, USA). PTEN antibody was prepared as previously described [50 (link)]. Anti-Trx1 and anti-rabbit IgG horseradish peroxidase-conjugated antibodies were purchased from Ab Frontier (Daejeon, Korea). Anti-tubulin antibodies were from Sigma-Aldrich. Immobilon Western chemiluminescence horseradish peroxidase substrate was purchased from Millipore Corporation (Billerica, MA, USA). NAP-5 Columns (Sephadex G-25 DNA Grade) were purchased from GE Healthcare (Little Chalfont, UK). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Capricorn Scientific GmbH (Ebsdorfergrund, Germany).