2.5. Cell Viability Assay

Cells were plated in 96 well plates at a density of 3 × 10⁴ cells per well. The day after, cell culture medium was replaced with medium containing different concentrations (ranging from 0.05 to 30 µM) of the drugs, or medium with equivalent DMSO concentration (vehicle) used as control. After 3 days, the medium was replaced, and the treatment was repeated. After 6 days, cell viability was analyzed using 3-(4,5-dymethylthiazol-2-yl)-2,5-dyiphenyltetrazolium bromide (MTT) assay, as previously described [24]. All experiments were performed in six replicates.

In vitro experiments (analysis of cell viability, cell cycle and apoptosis) were monitored for up to 6 days of drug incubation. We performed long-term treatments due to the slow doubling time (about 4 days) of the MTC cell lines, as previously reported [26]. Indeed, the effects of tested TKIs on cell viability were moderate after 3 days of incubation, as shown in Figure S1.