Figure 3

Depletion of Nup88 causes multipolar spindles. (A) Effects of Nup88 depletion on protein levels. Lysates of control siRNA-transfected HeLa cells and Nup88 siRNA-transfected HeLa cells at 72 h after transfection were analyzed by immunoblotting with the indicated antibodies. (B) Knockdown of Nup88 by siRNA leads to abnormal chromosome formation in mitosis. HeLa cells were transfected with a siRNA duplex against Nup88. After 72 h, the cells were stained with anti-Nup88 (green) and anti-Nup214 (red) antibodies. Chromatin was visualized using DAPI (blue). Confocal microscopy of Nup88 siRNA-treated cultured cells reveals a loss of nuclear envelope-associated Nup88 (white circle). Fluorescence intensity measurements performed on randomly selected interphase cells (Total) indicate a ≈70% reduction in the Nup88 level (± 5%; P < 0.001) in siRNA-treated cells versus control (Mock) cells. A larger mean reduction in the anti-Nup88 antibody fluorescence intensity of 80% (± 5%; P < 0.001) is observed when the measurements are restricted to cells containing multipolar spindles. Scale bar, 10 μm. The white asterisk indicates typical chromosome defects. (C) Knockdown of Nup88 by siRNA showed deprived spindle morphology and a significant increase in the frequency of multipolar spindles. The HeLa cells were stained with anti-Nup88 (green) and anti-α-tubulin (red) antibodies. Chromatin was visualized using DAPI (blue). Scale bar, 5 μm. (D) Mitotic HeLa cells were scored for multipolar spindles or cytokinesis defects. The data represents the means of three experiments in which 250 mitotic cells were scored at each time point.