In vitro tubulin polymerization assay

Tubulin polymerization was analyzed in vitro using a Tubulin Polymerization Assay kit (Cytoskeleton, Denver, CO). Briefly, 2 mg ml⁻¹ porcine tubulin was dissolved in buffer 1 (80 mM PIPES, 2 mM MgCl₂, 0.5 mM EGTA pH 6.9, 10 μM fluorescent reporter, 1 mM GTP, 15% glycerol). This solution was transferred to a pre-warmed 96-well plate and treated with 15 μM of the test compounds 6, 8 and 9. Colchicine and paclitaxel at 3 μM were employed as positive controls, while 0.1% DMSO treated-cells were employed as a negative control. Tubulin polymerization was monitored at 37 °C for 60 min using fluorescence microscopy. The reading speed was programmed at 1 cycle per min with excitation and emission wavelengths of 360 and 450 nm, respectively, using the Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific). The 100% polymerization value was defined as the area under the curve (AUC) of the untreated control.