Figure 3
Inhibition of microtubule polymerization by SSE15206. (A) SSE15206 inhibits microtubule polymerization in cells. A549 cells were incubated on ice for 30 minutes followed by incubation at 37 °C for 10 minutes in the presence of 1.25 μM and 2.5 μM SSE15206 or DMSO control. 100 ng/ml (0.33 μM) nocodazole was used as a control for microtubule depolymerization. Cells were analyzed by immunofluorescence using alpha-tubulin antibodies (green) and DAPI (blue). (B) SSE15206 induces the formation of aberrant mitotic spindles. A549 cells were treated with 0.5 μM and 1 μM SSE15206 for 24 hours, fixed and stained for Aurora-A (red), tubulin (green) and DNA (blue). (C) Increased aberrant mitotic spindles upon treatment with 0.5 and 1 μM SSE15206 (one-way ANOVA: F (1,4) = 246.8, p < 0.0001; post-hoc: SSE15206 0.5 μM vs. DMSO ****p < 0.0001, SSE15206 1 μM vs. DMSO ****p < 0.0001) (D). In vitro inhibition of tubulin polymerization by SSE15206. Polymerization of purified tubulin was measured in the presence of 5 μM and 25 μM SSE15206. DMSO was used as a solvent control while paclitaxel and nocodazole were used as controls for tubulin polymerization and depolymerization, respectively. (E) Docking of SSE15206 in the colchicine binding site of tubulin by Auto Dock Vina (upper panel). H-bonding interaction of SSE15206 with the beta subunit of tubulin in the docked structure (lower panel). (F) SSE15206 displaces colchicine in the competition assay. Different concentrations of SSE15206 were incubated with tubulin in the presence of 20 μM colchicine followed by measurement of fluorescence intensity. Colchicine displacement was quantified by relative fluorescent intensity (expressed as %, one-way ANOVA: F (4,10) = 84.5, p < 0.0001; post-hoc 25 μM vs. Control ***p < 0.001; 50 μM vs. Control ****p < 0.0001, 100 μM vs. Control ****p < 0.0001, 50 μM****p < 0.0001).
