Figure 4. Effects of Leiodermatolide on Polymerization/Depolymerization of Tubulin.

a) PANC-1 cells were treated for 24 hours with methanol (vehicle control), 100 nM leiodermatolide, 100 nM vinblastine or 1000 nM nocodazole. Cells were fixed and stained with an antibody against β-tubulin and counterstained with propidium iodide and subjected to confocal microscopy. Experiment was repeated 3 times and one representative experiment is shown. Leiodermatolide appears to induce depolymerization at this high 100 nM concentration. b) Purified bovine tubulin and c) bovine tubulin containing microtubule associated proteins (MAPS) were induced to polymerize by warming the tubulin to 37°C in the presence of 1, 10, 50 or 100 μM Leiodermatolide, 10 μM paclitaxel, 10 μM nocodazole, 10 μM vincristine, methanol or DMSO (vehicle controls) or buffer alone. Polymerization was enhanced by the presence of glycerol in the buffer. Polymerization was measured by recording turbidity every minute for 1 hour at 340 nm with a plate reader. Experiment was repeated 3 times and one representative experiment is shown. Leiodermatolide had no effect on tubulin polymerization or depolymerization on tubulin alone at any of the concentrations tested. Leiodermatolide had no effect on tubulin polymerization or depolymerization on tubulin with MAPS at 10 μM, but appeared to enhance polymerization at 100 μM when MAPS are present.