Fluorescence-based tubulin polymerization assay

Tubulin polymerization assay [25] kit BK011P (Cytoskeleton) was used according to the manufacturer’s instructions. Briefly, 5 μL aliquots of the test compounds solubilized in 10% DMSO were added (in triplicate) to the wells of a 96-well plate (OptiPlate-1536 F–Black, PerkinElmer). Then, 45 μL of the reaction mixture (2 mg/mL tubulin (> 99% pure), 80 mM PIPES, 0.5 mM EGTA, 1 mM GTP, 2 mM MgCl₂, glycerol 15% and 10 μM DAPI, pH 6.9) was added to each well, and the plate was immediately placed in a fluorimeter (Victor3, PerkinElmer). Fluorescence at 355_Ex/460_Em was measured every 90 s for 1 h. To determine IC₅₀, we applied 7 concentrations of the test-compounds ranging from 0.256 to 50 μM. The curves were normalized, and the percentage of inhibition was calculated by comparing the polymerization reaction at 25 min relative to vehicle control.