Figure 7: Influence of 17 on HeLa cells and microtubules.

(a) 17 induces mitotic arrest. HeLa cells were treated for 24 h with different concentrations of 17 or DMSO and nocodazole (Noc) as controls. Cells were then fixed and stained for DNA, the mitotic marker phospho-histone H3 and tubulin. High-content analysis was performed to determine the percentage of mitotic cells using the MetaMorph software. Data are shown as mean values (n=3)±s.d. (b) 17 impairs the microtubule cytoskeleton in HeLa cells. Cells were treated with 10 μM 17 or DMSO for 2 h. After fixation, cells were stained for tubulin and DNA using an anti-tubulin antibody coupled to FITC (green) and DAPI (blue), respectively. Scale bar, 20 μm. (c) 17 inhibits tubulin polymerization in vitro. Tubulin polymerization was monitored by means of increase of fluorescence intensity of DAPI on binding to microtubules at ex/em 340/460 nm. Data are representative of three independent experiments. (d) 17 inhibits the regrowth of microtubules in HeLa cells. Cells were treated with 10 μM 17 and DMSO for 2 h before incubation on ice for 1 h. Cells were rewarmed at 37 °C for given time intervals and then fixed and stained as described in b. Scale bar, 20 μm. Pictures shown are representative of three biological replicates. (e) 17 displaces BODIPY-FL-vinblastine from tubulin. Porcine brain tubulin was incubated with BODIPY-FL-vinblastine and different concentrations of 17 or vincristine as a control for 40 min at 25 °C. Fluorescence intensity was then monitored at ex/em 470/514 nm. Decrease in fluorescence indicates competition with BODIPY-FL-vinblastine for binding to the vinca-binding site. Data are shown as mean values (n=3)±s.d. and were normalized to DMSO.