Fig. 3. Depletion of N-WASP causes misalignment and missegregation of chromosomes. (A) Treatment with siRNA against N-WASP resulted in a marked decrease in N-WASP protein levels. (B) Control and N-WASPdeficient cells were synchronized by nocodazole for 16 h. The cells were harvested at different time points from nocodazole release and analyzed for DNA contents by FACS analysis. (C) N-WASP depletion causes misalignment of chromosomes at metaphase plate and abnormal chromosome congression. Percentages of cells with chromosome misalignment were indicated. N-WASP knockdown increased the incidence of misaligned chromosomes in metaphase cells. Data are means from 100 cells in three independent experiments. (D) N-WASPknockdown induced a delay in mitosis from prometaphase to anaphase. The percentage of cells in each mitotic stage was determined by DNA and microtubule staining. The bar graphs were generated using data from three independent experiments. (E) Prolonged mitosis in N-WASPknockdown cells. Video microscopy revealed that N-WASP-deficient cells were arrested in prometaphase. Exit from prometaphase was observed over a period of 140 min. (F) Depletion of N-WASP reduced the proliferation rate of cells. HeLa cells were transfected with control or N-WASP RNAi and then synchronized at early S phase with double thymidine block. After 60 min thymidine was released, during which time the cells were replated into a RT-CES device, and real-time measurement of well impedance was monitored. Dynamic cell proliferation was monitored as a cell index calculated from the impedance of each well measured every 30 min after re-plating. (G) Multinucleate cells with macro- and micronuclei and nuclear bridges were observed during interphase of N-WASPdeficient cells. Percentages of cells with aberrant multiple nuclei produced by N-WASP-knockdown were indicated. Data are means from 100 cells in three independent experiments.