Figure 2

Downregulation of DDX3 results in mitotic abnormalities, G2/M phase transition delay and increased cell death. (a) Mitotic-enriched cells were immunostained with anti-DDX3 (red), anti-α-tubulin (green) antibodies and DAPI (blue). Representative confocal images show the control mitotic cells with normal bipolar spindles (NB) and balanced chromosome segregation in metaphase (NB-M), anaphase (NB-A) and telophase (NB-T) while DDX3 knockdown promoted the abnormal mitosis such as chromosome misalignment (MisA) (arrow), chromosome segregation defect (arrowhead), lagging chromosome (Lag) and chromosome bridge (Bri) and multipolar mitosis (MuP-M, A, T). Scale bar = 5 μm. The percentage of aberrant mitosis in the control and DDX3-knockdown cells were analyzed. Data are shown as the average value ± S.D. calculated from three independent experiments. **P < 0.01; ***P < 0.001. (n), the number of cells analyzed. (b) (c). Histogram of cell cycle phase distribution in the control and DDX3-knockdown cells. DDX3 knockdown induced prolonged accumulation in G2/M phase at 12–16 hr after release from G1 (b) or S phase block (c). Cells were synchronized at G1 (b) or S (c) phase at 24 hr post-transfection and harvested at indicated time points after release. Harvested cells were stained with propidium iodide and analyzed by flow cytometry. Results are representative data of two independent experiments. (d) Western blot analysis showing the expression of γH2AX, Ser³⁴⁵-phosphorylated CHK1, CHK1 kinase and Tyr¹⁵-phosphorylated CDK1 in the control and DDX3-knockdown cells. Depletion of DDX3 caused an increase of γH2AX, a double strand DNA break marker, and activation of CHK1 kinase, which led to accumulation of phosphorylated CDK1 and prevented G2/M transition. Original images of western blots were presented in Supplementary Fig. S3. (e) DDX3 knockdown increased the distribution of cells in the sub-G1 peak. The proportions of sub-G1 phase population of the control and DDX3-knockdown cells are shown as the average value ± S.D. calculated from three independent experiments. **P < 0.01.