Calcusyn software program

The viability of the cells was measured with the TACS™ MTT cell proliferation assay (TREVIGEN systems) in accordance with the manufacturer's instructions. Briefly, cells were plated in 96-well plates at a concentration of 2000-20000 cells/100 μl/well. The next day, drugs were added at different concentrations; all experiments were performed in triplicate. The plates were incubated for six days at 37°C. Then, 10 μl of the MTT solution was added to each well and the plates were incubated for an additional four hours at 37°C. Then, a detergent solution (200 μl/well) was added and mixed thoroughly to dissolve the dark-blue crystals. The absorbance of the converted dye was measured spectrophotometrically with a microplate reader (Vmax, Molecular Devices, Sunnyvale, CA, USA) at 570 nm (test) and 650 nm (reference). Cell survival was calculated as the percentage of MTT inhibition as follows: % survival = (mean experimental absorbance/mean control absorbance) × 100 [46 (link)].
The possible synergistic effect of MLN8237 and IM was analyzed with the CalcuSyn software program (Biosoft, Ferguson, MO, USA), which is based on the Chou and Talalay method [24 (link)]. The combination index (CI) was calculated with CalcuSyn software. For the CI, a value >1 is defined as antagonism, equal to 1 as additivity, and <1 as synergy. The experiment was performed in triplicate.

The cytotoxicity of a test compound was determined, as previously described (Furuta et al., 2002 (link); Han et al., 2011 (link)) by measuring cell viability in the presence of various compound concentrations. Briefly, 100 μl MDCK cells (0.5 × 10⁵ cells/ml) were dispensed into wells of a 96-well plate and incubated at 37°C/5% CO₂ for 24 h. The test compound at the serial dilution in 100 μL of DMEM without serum was added to the cells. After culture for 72 h, cell viability was measured using the Cell Counting Kit-8 (CCK-8), which was purchased from Dojindo Laboratory (Kumamoto, Japan), and using a spectrophotometer (Ultra 384, Tecan, NC, United States). The percent cytotoxicity (=100% – % cell viability) was calculated, and CC₅₀ (half-maximal cytotoxic concentration) of the test compound was determined using the Calcusyn software program (Biosoft, Ferguson, MO, United States) (Chou and Talalay, 1984 (link)).

Combination analysis of treatment with foretinib and lapatinib was performed using the Calcusyn software program (Biosoft, Cambridge, UK), which calculates a combination index, according to Chou and Talalay-derived equations [36 (link)]. CI <1, =1, and >1 represent synergistic, additive, and antagonist effects, respectively.