Inhibition of CPAP-tubulin interaction prevents proliferation 
of centrosome amplified cancer cells (2018)
Figure 7

Figure 7: CCB02 effects differ from the effects of known tubulin binding agents.

(A) Bar graph quantifies relative intensity of PCM proteins at interphase centrosomes of cells treated with vehicle, CCB02, and known tubulin binders. Two centrosomes containing MCF10A (-Dox, two centrosomes) cells were used. Elevated PCM proteins intensities were detected only with CCB02 compared to vehicle treated cells. However, a slight increase in PCNT was observed with Docetaxel treatment. Number of centrosomes tested, n=100 (vehicle); n=100 (for CCB02, Taxol, Bacatalin III, Docetaxel and Vinblastine). (N)=3. Error bars, mean ± SEM. Ordinary Two-way ANOVA test **P < 0.001, ***P < 0.0001.

(B) CCB02 but not conventional tubulin binders (Taxol, Bacatalin III, Docetaxel and Vinblastine; concentrations used 30nM) causes activation of extra centrosomes and prevents them from clustering. MCF10A cells with extra centrosomes (+Dox, extra centrosomes) were used. In contrast to CCB02-treatment, interphase centrosomes of tubulin binders-treated cells consistently display clustered centrosomes. Note, disrupted mitotic spindles were observed in tubulin binders-treated cells due to their general toxicity to microtubules. Cells were stained with CPAP (green), Cep152 (magenta) and microtubules (α-tubulin, red). Scale bar, 2µm. Three independent experiments (N)=3 with each at least 100 cells were examined per treatment.

(C) Bar graph quantifies percentages of cells exhibiting multipolar spindles in interphase and mitosis of vehicle, CCB02 and conventional tubulin binders-treated cells. At least 150 cells were analyzed for each independent experiment (N)=3. Error bars, mean ± SEM. Ordinary Two-way ANOVA test **P < 0.001.

(D) Kymographs (i) and averaged microtubule polymerization velocities mediated by vehicle and CCB02. Graph below represents the microtubule growth velocities mediated by vehicle (red) and CCB02 (blue) during microtubule end-tracking assay (ii). CCB02 does not alter the microtubule growth although there is a slight increase in growth at 5µM. At least 75 filaments were analyzed for each condition. The error bars represent mean ± SEM of three independent experiments (N)=3.

(E) Snap-shot images show the effect of CCB02 on microtubule dynamics in two-centrosome containing MCF10A cells. The microtubule was stained with SiR-tubulin and +end tip of microtubule was marked with EB3-EGFP. A 5µM CCB02 did not alter localization and dynamics of EB3 at the microtubules indicating that the CCB02 does not affect microtubule dynamics (For details, refer to Movies-EV4-EV5). At least, 50 cells were analyzed for each condition. Scale bar, 2µm.

(F) Box plots represent the speed (µm/sec) (i), lifetime (sec) (ii), and length (µm) (iii) of growing microtubule tracks in MCF10A cells treated with 1, 2 and 5µM of CCB02. At least, 60 filaments were analyzed from each cell from a total of 30-40 cells. (N)=3. Error bars in boxplots mean ± SD. Ordinary One-way ANOVA test was performed. Boxes in all plots show the growing microtubule parameters such as speed (µm/sec), lifetime (sec) and length (µm/sec). Horizontal lines in boxes show the median value. Whiskers at both extremes of boxes indicate minimum and maximum values.