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. Author manuscript; available in PMC: 2018 Apr 21.
Published in final edited form as: ACS Chem Biol. 2017 Feb 22;12(4):1038–1046. doi: 10.1021/acschembio.6b01040

Figure 4. Effects of Eg5 inhibitors on microtubule stability and spindle integrity.

Figure 4

(a) Effects of inhibitors on MT depolymerization following taxol washout. Cy5-labeled, taxol-stabilized MTs were bound to coverslips using a kinesin-1 rigor mutant, and taxol was washed out to induce MT depolymerization. For ATP and MON+ATP, Eg5 was presented at 40 nM, whereas for the strong-binding species BRD, AMPPNP, and Apo, only 5 nM Eg5 was used, emphasizing the potency of Eg5 in stabilizing MT under these conditions. All samples included 1 vol% DMSO. All data are shown (points) along with the mean (line) and fit to a normal distribution (curve). (b) Spindle collapse assays in the presence of varying inhibitors. Representative images of RPE-1 cells are shown, immunostained for tubulin (green), centrin (magenta), and DNA (blue). Scale bar: 5 μm. Quantification of spindle integrity. In the control group (no Eg5 inhibitor), the spindle retained its bipolar geometry and no monopolar spindles were observed (N = 89). In the presence of the L5 inhibitor STLC, 69 ± 9% of spindles collapsed to monopole phenotype (mean ± s.d., N = 94). In contrast, in the presence of the rigor inhibitor BRD9876, 1 ± 1% spindles were monopolar (mean ± s.d., N = 81). Eg5 localization under different conditions is shown in Figure S6.