Transforming growth factor tgf β

MDA-MB-231 and MCF7 cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MDA-MB-468 cells were kindly provided by Dr C. Gilles (University of Liège, Belgium). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza) containing 10% fetal bovine serum (FBS, ThermoFisher Scientific) and 2 mM L-glutamine (Lonza). For cell signaling analysis, cells were cultured in DMEM with L-glutamine and without FBS. L-carnosine (C9625), aminoguanidine (396494) and methylglyoxal (MG, M0252) were from Sigma. We excluded the presence of significant formaldehyde contamination (< 3%) in MG (lot #BCBQ9416V) by nuclear magnetic resonance (NMR) analysis. Transforming growth factor (TGF)β (#10021) was from Peprotech. U0126 (S1102) was from Selleckchem. Anti-argpyrimidine antibody (mAb6B) specificity has been previously confirmed by competitive enzyme-linked immunosorbent assay (ELISA) and shown to not react with other MG-arginine adducts [13 (link)].

Dulbecco’s modified Eagle medium (DMEM), minimum essential medium-alpha (α-MEM), Dulbecco’s modified Eagle medium:nutrient mixture F-12 (DMEM/F-12) without phenol red, Dulbecco’s phosphate buffered saline (PBS), fetal bovine serum (FBS), Hank’s balanced salt solution (HBSS), antibiotic-antimycotic mixture containing 100 U/mL penicillin and 100 U/ mL streptomycin, Opti-MEM, Geneticin (G418), and 0.25% trypsin-EDTA were purchased from Gibco BRL (Grand Island, NY, USA). Recombinant human chemerin (Glu21-Ser157, with an N-terminal Met), mouse soluble receptor activator of nuclear factor kappa beta (RANK) ligand (RANKL), and macrophage colony-stimulating factor (M-CSF) were purchased from R&D Systems (Minneapolis, MN, USA). Anti-human E-cadherin (sc-8426), β-catenin (sc-1496-R), vimentin (sc-6260), lamin B (sc-6216), RANKL (sc-377079), osteoprotegerin (OPG; sc-71747), Smad2/3 (sc-133098), and GAPDH (sc-32233) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Transforming growth factor (TGF)-β and insulin-like growth factor (IGF)-1 were purchased from Peprotech (Rocky Hill, NJ, USA). Histopaque-1083, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents used in this study were of analytical grade.

SVF and PBMC from obese patients and PBMC from LC were thawed, washed in RPMI, counted and incubated with carboxyfluorescein succinimidyl ester (CFSE) (CellTrace CFSE Cell Proliferation Kit, Life Technologies, ThermoFisher). After labeling, 300 000 cells were plated in round-bottom, 96-well plates coated with anti-CD3 (1 µg/mL, Miltenyi Biotec) to assess cell proliferation and cytokine production. In some conditions, cells were coincubated with transforming growth factor (TGF)-β (5 µg/mL, PeproTech) to assess T cell susceptibility to suppression. After 96 hours of incubation at 37°C, cells were stimulated with PMA/ionomycin following the protocol for intracellular cytokines detection. Per cent suppression was calculated using the following formula: ((% of proliferative T cells alone – % of proliferative T cells treated with TGF-β)/% of proliferative T cells alone) × 100.