Figure 4.
MDs bind irreversibly to β-tubulin. A, tubulin (3 mg/ml) incubated with 30 μm colcemid or 30 μm SKLB028 for 1 h. Then tubulin part and filtrate part were separated with an ultrafiltration method. Compounds in tubulin part and filtrate part were detected by HPLC. Colcemid (30 μm) or SKLB028 (30 μm) dissolved in 200 μl of CH3OH were also detected by HPLC as controls. This experiment was repeated three times. B, high concentrations (10 μm) of SKLB028 or colcemid were co-incubated with HCT-8/V cells for 8 h, and then compounds were extensively washed for three times. The cells were further cultured to 72 h. At 0-, 8-, 24-, 48-, and 72-h points, tubulin morphology was analyzed by immunofluorescence. Bar, 20 μm. Tub, tubulin; AU, absorbance unit.