2.7. In Vitro Tubulin Polymerization Assay

Tubulin polymerization kinetics was assessed in vitro using a tubulin polymerization assay kit (Cytoskeleton #BK011P, Denver, CO, USA). In brief, 2 mg/mL 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) to reach a concentration of 10 mg/mL. The tubulin solution was then transferred to a pre-warmed 96-well plate containing the IC₅₀ dose of SIMR1281 along with 0.5 µM CaCl2 and 3 µM paclitaxel. Tubulin polymerization was monitored as a fluorescence signal at 37 °C for 60 min at an excitation wavelength of 360 nm and an emission wavelength of 450 nm. Reading was carried out using the Multi-skan go (Thermo Fisher Scientific, Waltham, MA, USA).