Pyruvate

U251 and U87 cell lines derived from human GBM were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM, Biowest, Nuaillé PDL, France) supplemented with 10% fetal bovine serum (FBS), 0.1 mM non-essential amino acids, 1 mM pyruvate, and 1 mM antibiotics (Biowest) in a humidified atmosphere with 5% CO₂ at 37 °C. Before treatments, cells were seeded in DMEM no phenol red (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% hormone-free FBS (Thermo Fisher Scientific), 0.1 mM non-essential amino acids, 1 mM pyruvate, and 1 mM antibiotics (Biowest). Cells were treated with 10 nM E2 (Sigma-Aldrich, St. Louis, MO, USA), 10 ng/mL TGF-β (PeproTech, Rocky Hill, NJ, USA), and vehicle (0.01% cyclodextrin + 0.0001% BSA).

HeLa cells, derived from human cervix cancer, were grown in DMEM (Dulbecco’s modified Eagle’s medium) (Biowest, Miami, FL, USA) supplemented with 10% FBS (Gibco, Paisleg, UK), 1mM of L-glutamine (Sigma Aldrich, St Louis, MO, USA), 1mM sodium of pyruvate (Biowest, Miami, FL, USA), and 100 U/mL of penicillin––streptomycin (Euroclone, Devon, UK). JetPEI transfection reagent (Polyplus Transfection, Illkirch, France) was used, following the manufacturers’ protocol.

U87 and U251 (ATCC, VA, USA) cells derived from human glioblastomas were cultured in Dulbecco's modified eagle medium (DMEM) with phenol red, supplemented with fetal bovine serum (FBS) (10%), pyruvate (1 mM), glutamine (2 mM), and nonessential amino acids (0.1 nM) (Biowest, Nuaillé, FRA); the culture was maintained at 37°C, under 95% humidity/5% CO₂ atmosphere. Cells were grown until reaching a 70–80% confluence.