Dmem high glucose with l glutamine

HCT116 cells were purchased from American Type Culture Collection (ATCC–Manassas, VA, USA). High glucose Dulbecco’s Modified Eagle’s medium (DMEM/HIGH Glucose) with L-Glutamine was purchased from Euroclone (MI, Italy), penicillin–streptomycin solution for cell culture was purchased from Gibco (NY, USA). Fetal bovine serum (FBS) was purchased from Thermo Scientific (HyCloneTM). Crystal phosphate buffer saline (PBS) (0.01 M Phosphate buffer, 0.0027 M KCl e 0.14 M NaCl, pH 7.4 at 25 °C) was purchased from Bioline (TR, Italy).
Deuterium oxide (D₂O, 99.8%D) was obtained from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade.

HeLa cells were grown in DMEM high glucose with L-glutamine (EuroClone) supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. The day before transfection, 0.15 × 10⁶ cells/well were seeded in antibiotic-free medium in a 6-well plate. Cells at 25–30% confluence were transfected with 50 nM siRNA (control pool of scramble oligos and NF-YB siRNA J-010002-08-0002, ON-TARGETplus, Dharmacon) using 3.75 μL Lipofectamine 3000 Reagent (ThermoFisher) in 1.5 mL final volume of Optimem (ThermoFischer) per well. 16 hours post-transfection, cells were detached by trypsin treatment, pooled and split in new wells. 72 hours post-transfection cells were harvested for protein extracts and RNA preparation. Three independent inactivation experiments were performed. The RNAs were isolated using TRI-reagent (Merck) and further purified with RNeasy Mini Kit (Qiagen) following the RNA clean-up protocol. RNAs were then quantified with Nanodrop and RNA integrity assessed with Agilent Tapestation. Total protein extracts were prepared in RIPA buffer and used for Western blotting. The membrane was probed with primary antibodies and secondary HRP-conjugated secondary antibodies (Sigma Aldrich). Primary antibodies: anti-NF-YA (G2, Santa Cruz Biotechnologies), anti-NF-YB (GeneSpin), and anti-Vinculin (Sigma Aldrich) as loading control.