Figure 4.

(a) Cell-free tubulin polymerization assay in vitro. Purified tubulin was used to test the ability of BPT to inhibit tubulin polymerization in vitro. The assay measures the increase in optical density as a result of tubulin assembly or polymerization. Nocodazole and paclitaxel were used in the assay as a known inhibitor and enhancer of tubulin polymerization. BPT was tested at four different concentrations that show inhibition of cell growth in vitro. The change in V_max value was used as an indicator of tubulin/ligand interactions. The polymerization curves indicate 0.5 , 1, 2.5 and 5 μM of BPT reduced the V_max value from 19 mOD/min (control) to 12.5, 9.2, 3 and 0.5 mOD/min, respectively, in a dose-dependent manner. The curves shown represent the average of three independent experiments. (b) Inhibition of tubulin polymerization and enhancement of tubulin depolymerization in live cells. The tubulin polymerization assay was performed in A549 (whole cells) after 30 min compound treatment at the concentrations indicated in the figure. Supernatant and pellet represent unassembled and assembled tubulin, respectively. Tubulin polymerization is detectable by the increase of tubulin in pellet and its disappearance from supernatant. The western blots show dose-dependent inhibition of tubulin polymerization after the simultaneous treatment of paclitaxel and BPT that resulted in the accumulation of unassembled tubulin in supernatant. BPT also acts as an enhancer for tubulin depolymerization in a dose-dependent manner when paclitaxel-stabilized tubulin was subjected to BPT treatment for 30 min