PrestoBlue cell viability assay

The transformed 3T3 cells expressing each FGFR mutant were cultivated in 96-well plates in DMEM-F12 medium (100 µL of culture medium per well) with 1.5% FBS and FGFR inhibitor at concentrations ranging from 0.1 nM to 10 µM for 5 days. The transformed Ba/F3 cells expressing each FGFR mutant were cultivated in 96-well plates in RPMI 1640 medium with 20% FBS and FGFR inhibitor at concentrations ranging from 0.1 nM to 10 µM for 5 days. Subsequently, 10 μL of PrestoBlue (Thermo Fisher Scientific) was added to the plates, and the fluorescence was measured after 3 h of incubation (excitation 530 nm, emission 590 nm) at 0.1 s. The fluorescence intensities of wells without cells were used as negative controls, and dose-response curves were fit the observed cell viabilities using the “drc package” in R language⁵⁸. The three-parameter sigmoidal function LL2.3 was used with the following settings: y0 (response without drug) = 0, robust = “mean”, method = “Nelder-Mead”. The IC₅₀ was defined as the inflection point on a dose-response curve.