Fig. 5. Mdivi-1 associates with tubulin and inhibits tubulin polymerization.

a Mdivi-1 quenched the intrinsic tryptophan fluorescence of tubulin. The effect of mdivi-1 at indicated concentrations on intrinsic tryptophan fluorescence of tubulin was measured as described. The excitation wavelength was 295 nm. The effect of tubulin polymerization inhibitor vinblastine was also measured as a positive control. Means ± SD from three independent experiments is shown. b The mean reductions of tryptophan fluorescence at 355 nm (ΔFL355) induced by various concentrations of mdivi-1 from (a) are expressed as a double reciprocal plot, yielding a regression line with R² value of 0.99. c Mdivi-1 inhibited tubulin polymerization in vitro. In vitro tubulin polymerization was carried out in the absence or presence of various concentrations of mdivi-1, and the level of polymerized tubulin was estimated by the absorbance at 340 nm (A340). The effects of taxol and vinblastine were also measured as positive and negative controls, respectively. The means of the three experiments are shown. d Mdivi-1 inhibited tubulin polymerization in arrested abnormal mitotic cells. A cellular tubulin polymerization assay was performed as described, and western blot was used to analyze the level of monomer tubulin in the supernatant (S) and polymerized tubulin in the pellet (P) of the lysate. The numbers underneath the blot indicate the mean percentages (two experiments) of each tubulin fraction relative to the total tubulin content (monomer + polymerized tubulin).