Dimethyl sulfoxide (dmso)

Whole blood (~350 mL) was collected from both male and female healthy adult donors via antecubital venipuncture into BD vacutainer, and Sodium Heparin blood collection tubes (BD, Franklin Lakes, NJ). Blood was diluted 2-fold in calcium- and magnesium-free phosphate-buffered saline (PBS pH: 7.2; Fisher Scientific, ​​Waltham, MA) within 30 min of collection. A single-step density gradient centrifugation was performed to isolate peripheral blood mononuclear cells (PBMCs) by slowly layering diluted blood (30 mL) over 15 mL of Lymphoprep™ (STEMCELL Technologies, Vancouver, Canada) contained within either a 50 mL SepMate™ (STEMCELL Technologies) or a 50 mL conical tube. Stepwise centrifugation at 1,200 x g for 10 min at room temperature with slow deceleration was performed. The buffy coat interface containing PBMCs was collected into 50 mL conical tubes and pelleted via centrifugation (1,400 RPM for 10 min). Residual red blood cells were removed with treatment with ACK Lysing Buffer (Gibco, Waltham, MA). PBMCs were then washed twice with 1X PBS, pelleted as above, and cryopreserved at -80°C in 90% (v/v) fetal bovine serum (FBS; R&D Systems, Minneapolis, MN) and 10% (v/v) dimethyl sulfoxide (Corning, Corning, NY) in cryovials labeled with each donor’s unique sample identification number, followed by transfer to liquid nitrogen storage within 24 h.

DIM (cat. # D3232, LKT Laboratories, St. Paul, MN, USA), RG-7388 (cat. # 21532, Cayman, Ann Arbor, MI, USA), and Nutlin-3a (cat. # 44151, Millipore, Billerica, MA, USA) were dissolved/diluted in dimethyl sulfoxide (DMSO, Corning, Manassas, VA, USA) and applied to the cell culture to reach the indicated concentrations.

Ethanol (95%) (BDH laboratory-England), Deionized water, Petroleum ether (BDH laboratory-England), Ethyl acetate (BDH laboratory-England), Rotary evaporator (Heidolph-Germany), Separating funnel. Dulbecco’s modified eagle medium (DMEM) (caisson labs, USA), fetal bovine serum (FBS) (Hyclone, South America) penicillin–streptomycin (Hyclone, US), Phosphate buffered saline (PBS) (Oxoid, England), (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) (Bioworld, USA), Dimethyl sulfoxide (DMSO), syringe filters 0.2 µm, Eppendorf tubes, T-flask 25 cm² (corning, USA), hemocytometer, 96 well plates (corning, USA), petroleum ether dilutions (100 µg/mL, 200 µg/mL and 300 µg/mL), ethyl acetate dilutions (100 µg/mL, 200 µg/mL and 300 µg/mL).