In vitro tubulin polymerization assay

Tubulin polymerization assay was conducted by using CytoDYNAMIX screen 03 kit (Cytoskeleton Inc., Denver, CO, USA). All steps were followed by manufacturer's protocol. Before preparing tubulin polymerization (TP) buffer, a 96 well-plate was pre-warmed in the Spectrophotometer at 37°C. TP buffer (including General Tubulin Buffer, Tubulin Glycerol Buffer and GTP), 85 μL/well and the test drugs (2 μL/well) were added to the pre-warmed 96-well plate, respectively. Here, DMSO was used for control, paclitaxel and vincristine for positive controls. Tubulin proteins were immediately added to the mixture (30 μL/well). The absorbance, 340 nm, were recorded every 1 min for 30 min at 37°C by ELISA reader (SpectraMAX Plus; Molecular Devices Inc., Sunnyvale, CA, USA).