Figure 4.

Rigosertib Inhibits Cell Division and Microtubule Growth in Cells

(A–D) Imaging of RPE1 CRISPRa (A and B) or CRISPRi (C and D) cells expressing either a control sgRNA or a KIF2C-targeting sgRNA after treatment with indicated concentrations of rigosertib.

(A) Representative images of fixed cells immunostained for tubulin (red). DNA was visualized with DAPI (blue).

(B) Mitosis durations of cells determined by fluorescence time-lapse microscopy. At least 60 cells were scored for each condition over 3 independent experiments. ^∗p < 0.05; one-tailed paired t test.

(C and D) Spinning disk confocal microscopy of cells expressing EB3-GFP.

(C) Representative maximum intensity projections of 50 time points (50 s). See also Movie S1.

(D) Quantification of microtubule growth speeds (left). Number of cells and microtubules scored over 3 independent experiments for each experimental condition: no rigosertib: control sgRNA (12, 177), KIF2C sgRNA (9, 152); 75 nM rigosertib: control sgRNA (7, 113), KIF2C sgRNA (8, 128). Quantification of microtubule-cortex contacts (right). Number of cells scored over 3 independent experiments: no rigosertib: control sgRNA (21), KIF2C sgRNA (18); 75 nM rigosertib: control sgRNA (29), KIF2C sgRNA (18).

All data represent mean ± SEM. Scale bars, 5 μm. See STAR Methods for details. See also Figure S4.