To test whether coumarin-30 interfered with any aspects of normal microtubule behavior, we examined its effect on the dynamics of individual microtubules in vitro (Fig. ), using differential interference contrast microscopy. Coumarin-30 exerted a concentration-dependent inhibition of microtubule dynamics, in a manner analogous to the well-known colchicine site binder, nocodazole, but distinct from the microtubule stabilizer, paclitaxel (Fig. ). A twofold suppression of the microtubule assembly rate and a statistically significant decrease in the microtubule catastrophe frequency were achieved at a submicromolar concentration of coumarin-30 (IC50 = 0.7 ± 0.1 μM) (Fig. ). relationship of microtubule dynamics. In this panel, time is represented along the y-axis, while space (position along the microtubule) is represented along the x-axis. The boundary between the stable microtubule seed and the dynamic extension of the microtubule is marked with a red dotted line. Controls 1 and 2 show microtubule dynamics in the absence of coumarin-30 at the beginning and end of the experiment to ensure that normal microtubule dynamics is fully restored after drug is washed out of the microscopy flow chamber after each test. 1 μM nocodazole (noc.) and 1 μM paclitaxel (pac.) are used as positive controls. Scale bar 5 μm. d) Quantification of the microtubule growth rate and catastrophe frequency in the presence of 0.5 μM nocodazole, 0.02 μM paclitaxel or various coumarin-30 concentrations. Each point represents the mean of at least N = 3 independent experiments. Statistical significance was assessed using an unpaired t-test with Welch correction. ns -not significant; *, **, **** -p-value (twotailed) < 0.05, <0.01, < 0.0001, respectively. e) Confocal microscopy images of A549-cells, fixed after 24 hours of treatment with DMSO, 1 μM nocodazole, 1 μM paclitaxel or 0.67 μM coumarin-30. Immunofluorescent staining with Alexa Fluor 594 anti-α-tubulin antibodies is pseudo-colored green, DNA is visualized with Hoechst staining, pseudo-colored magenta. Scale bars are 5 μm. See Fig. and S2 for more details. f) Histograms showing the distribution of A549 cells by DNA content after 48 hours of treatment with efficient concentrations of coumarin-30. Each displayed histogram is based on one representative experiment. g) Quantification of the fraction of cells in the G2/M phase in the population as a function of coumarin-30 concentration, based on the distribution of DNA content. Data points represent the means of N = 3 independent experiments. The upper grey zone indicates the mean ± standard deviation for cells treated with 0.3 or 1 μM nocodazole, used as a positive control. The lower grey zone indicates the mean ± standard deviation for cells treated with DMSO, used as a negative control. h) Quantification of accumulation of mitotic cells after 12 hours incubation with coumarin-30, nocodazole or DMSO in three cell types, based on phase microscopy of live cells. Data are mean ± standard deviation based on N = 3 independent experiments.