Preparation of Pinocembrin, Doxorubicin, and Dexrazoxane

Stock solutions of Pinocembrin (MW: 256.25 g/mol) (Pin, 10 mM, BioPharm™, New Zealand) and Dexrazoxane (MW: 268.269 g/mol) (Dex, 1 mM) (Sigma-Aldrich, St Louis, MO, USA) were prepared in Dimethyl sulfoxide (DMSO, Sigma-Aldrich, Saint Louis, MO, USA). DMSO concentration was kept below 0.0001%. Doxorubicin hydrochloride (MW: 579.98 g/mol) (Dox, 1 mM) was prepared in tissue culture (TC) grade water. Thereafter, final concentrations of 1 µM (Pin), 20 µM (Dex) and 2 µM (Dox) were prepared in Dulbecco’s modified Eagle medium without phenol red (DMEM, Lonza, Walkersville, MD, USA) supplemented with 2% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA) and then filter sterilized using 0.22 µM syringe filter systems prior to commencing treatment. Doses for Dox and Dex were derived from the clinically recommended therapeutic ratio of 1:10 (Rharass et al., 2016), while the concentration used for Pin was extrapolated from our preliminary dose response findings.