FIGURE 4.

Chromosome lagging, abnormal chromosome congression, and defective mitosis after Tpr siRNA treatment. A, effects of Tpr siRNA treatment on protein levels of Tpr-associated proteins. Lysates of control siRNA-transfected HeLa cells (control) and of HeLa cells, 72 h after transfection with Tpr siRNAs (Tpr RNAi) were analyzed by immunoblotting with the indicated antibodies. The same membrane was stripped and reprobed with anti-α-tubulin (as loading control). B, Mad1 was significantly reduced in the Tpr-depleted samples, whereas dynein and dynactin (p150) were not affected. C, representative images of asynchronous HeLa cells, transfected with either siRNA duplex against Tpr. 72 h after transfection, cells were stained with anti-Tpr antibody (green) and anti-dynein (red) and analyzed by confocal laser microscopy. Chromatin was stained with DAPI (blue). Scale bar, 5 μm. Tpr still displayed normal nuclear rim staining in a few HeLa cells. Mitotic dynein was still partially localized at kinetochores 72 h after transfection with Tpr-specific siRNAs (asterisks). White arrows indicate cells with chromosome defects when Tpr was completely depleted. D, schedule of collecting mitotic HeLa cells after siRNA Tpr depletion. E–G, representative images of mitotic HeLa cells, transfected with either siRNA duplex against Tpr. 72 h after transfection, cells were stained with (E) anti-dynein (red), (F) anti-Mad1 (red), (G) anti-Mad2 (red) antibodies, anti-Tpr antibody (green), and analyzed by confocal laser microscopy. Chromatin was stained with DAPI (blue). Scale bars, 5 μm. Abnormal chromosome congression, multipolar phenotypes, and chromosome lagging were often found. White arrows indicate cells with chromosome lagging. Mitotic Mad1 signal was reduced at the kinetochores 72 h after transfection with Tpr-specific siRNAs.