Calcusyn 2

Drug toxicity was determined as previously described (16 (link)). Briefly, cells were seeded in 96-well plates (3,000 cells/well) in triplicate and were treated with a range of concentrations of the indicated drugs, as depicted in Fig. 1, at 37°C for 72 h. Controls were treated with vehicle (either PBS or DMSO). Drug toxicity was assessed after staining with MTT (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) by measuring the production of formazan, which is directly proportional to the number of viable cells. Briefly, to each well of a 96-well plate containing 100 µl medium, 10 µl MTT solution (5 mg/ml MTT in PBS) was added and incubated at 37°C for 2 h. Solubilization of formazan was achieved following the addition of 100 µl solubilization solution [40% dimethylformadide v/v in 2% glacial acetic acid v/v, to which 16% sodium dodecyl sulphate (pH 4.7) had been added w/v] and reading optical density at 570 nm. For drug combination experiments, a combination index (CI) was calculated using CalcuSyn 2.0 software (Biosoft, Cambridge, UK) based on the Chou and Talalay method (17 (link)). CI values between 0.1 and 0.9 refer to different grades of synergism, values between 0.9 and 1.1 refer to additive effects, whereas values >1.1 refer to antagonistic effects.