Figure 5: Rigosertib interacts with tubulin in RMS cells, which destabilizes microtubules and induces mitotic spindle defects.
(A) Treatment of RD or SKNAS with 2 μM rigosertib for 24 hours decreases acetylation of α-tubulin as determined by quantitative capillary immunoassay. (B) RD (top) or SKNAS (bottom) were treated with increasing concentrations of rigosertib for 4 hours prior to lysis and separation of polymerized (P) and soluble (S) tubulin by centrifugation. The pellets were resuspended in a volume equal to that of the soluble fraction. Equal volumes of P and S for each condition were subjected to SDS-PAGE and immunoblot for α-tubulin. (C) Treatment of RD (top) or SKNAS (bottom) with 250 nM rigosertib for 24 hours induces mitotic spindle defects in unsynchronized cells, as determined by immunofluorescence staining of α-tubulin (mitotic spindles) and pericentrin (centrosomes). Representative images are shown. Boxed insets are shown as zoomed images at right and display multipolar mitotic spindles and other spindle defects.