Figure 3
DDX3 specifically prevents multipolar mitosis by clustering or inactivating extra centrosomes. (a) Representative confocal images showing bipolar and multipolar mitosis. Cells with more than two centrosomes can undergo multipolar mitosis or bipolar mitosis through inactivation (arrowheads) or coalescence (arrow) of the extra centrosomes. Cells were immunostained with γ-tubulin (green) and DNA (blue). Images are shown as the maximum projections of confocal Z stacks. Scale bar = 5 μm. (b) Depletion of DDX3 impaired the centrosome inactivation/coalescence and promoted multipolar mitosis in HCT116 cells. Confocal images of control or DDX3-knockdown mitotic cells classified as normal bipolar mitosis (N), pseudo-bipolar mitosis by centrosome inactivation/clustering (I/C) or multipolar mitosis (M). Each typical mitotic cell is shown at higher magnification in the right column. Cells were immunostained with DDX3 (red), γ-tubulin (green) and DNA (blue). Images are shown as the maximum projections of confocal Z stacks. Scale bar = 5 μm. The proportions of bipolar mitosis (two centrosomes), pseudo-bipolar mitosis (more than two centrosomes) and multipolar mitosis in the control and DDX3-knockdown cells were statistically analyzed (right panel). Data are shown as average value ± S.D. calculated from two independent experiments. *P < 0.05; **P < 0.01. (n), the number of cells analyzed. (c) Reintroduction of shDDX3-resistant Flag-DDX3 rescued the centrosome inactivation/coalescence and prevented multipolar mitosis in the DDX3-knockdown HCT116 cells. Cells were immunostained with DDX3 (red), Flag (blue), γ-tubulin (green) and DNA (gray). Images are shown as the maximum projections of confocal Z stacks. Scale bar = 5 μm. The proportions of mitosis in the DDX3-knockdown and DDX3 overexpressed DDX3-knockdown (si+FDR) cells were statistically analyzed (right panel). Data are shown as average value ± S.D. calculated from two independent experiments. *P < 0.05. (n), the number of cells analyzed.
