Flubendazole inhibits tubulin structure, polymerization, and function. (A) Flubendazole (100μM) and vinblastine (10μM) were incubated with bovine tubulin (1.5μM), and the conformational changes were monitored spectrophotometrically by measuring the decrease in the number of reactive cysteine residues at an absorbance of 412 nm as described in “Methods.” A representative figure is shown. (B) Flubendazole (100μM), colchicine (6μM), and taxol (6μM) were incubated with bovine tubulin (1.8 mg/mL), and the effects on polymerization were monitored spectrophotometrically by measuring turbidity at 340 nm as described in “Methods.” A representative figure is shown. (C) Tubulin (5μM) was incubated for 30 minutes with 100μM vinblastine, 100μM flubendazole, or buffer control. After incubation, colchicine (10μM) was added and incubated for 60 minutes. Fluorescence of the tubulin-colchicine complex was measured with excitation and emission wavelengths of 360 nm and 430 nm, respectively. Reduced fluorescence indicates binding at the colchicine site. *P < .01 (analysis of variance, Bonferroni post hoc). A representative figure is shown. (D) PPC-1 cells were treated with vehicle control (i) or 1.0μM flubendazole (ii) for 24 hours and stained with DAPI and an anti–α-tubulin Alexa Fluor 488-nm antibody. Images were captured using an Olympus Fluorview confocal microscope at room temperature. Representative confocal micrographs (original magnification ×40) are shown. (E) HeLa cells were grown to confluence and a wound created on the cell monolayer using a 200-μL pipette. Cells were treated with increasing concentrations of flubendazole and imaged every 2 hours for 8 hours. Wound healing was measured as described in the supplemental Methods. Representative data are shown and are presented as percentage wound recovery. *P < .05 (analysis of variance, Bonferroni post hoc).