ELR510444 arrests RCC cells in mitosis and depolymerizes microtubules.

<p>(A) RCC cells were treated with 10 and 30 nM ELR510444 for 24 h. Cell cycle distribution was determined by PI-FACS analysis. Representative histograms and the percentage of cells in each phase of the cell cycle are shown for each experimental condition. (B) RCC4 cells were plated on chamber slides and treated for 24 h with the indicated concentrations of ELR510444 or 100 nM vincristine. Cells were stained with a β-tubulin antibody and microtubules were visualized using an Alexa Fluor 488-conjugated secondary antibody. Nuclei were counterstained using DAPI. The percent microtubule depolymerization was determined at each concentration visually in 100 cells. Representative images are shown. (C) ELR510444 interacts with the colchicine-binding site of β-tubulin. RCC4 cells were pre-treated with the indicated concentrations of ELR510444 for 2 h. 100 µM EBI was then given for an additional 1.5 h. Cells were harvested for immunoblotting and blots were probed with a β-tubulin antibody to detect native β-tubulin and EBI: β-tubulin adducts.