Figure 3.

Depletion of hSpindly induces chromosome misalignment and mitotic delay. (A) Mitotic indices were determined for HeLa S3 cells after treatment for 48 h with GL2 (control) or two independent siRNAs targeting hSpindly (hSpindly-1 and hSpindly-2). Bar graphs show the results of three independent experiments (>100 cells each) and error bars indicate SD. The bottom panel shows Western blotting of cells treated for the indicated times with the above-mentioned siRNAs. Membranes were probed with anti-hSpindly antibody and α-tubulin is shown as loading control. (B) Cells treated with GL2 or hSpindly-2 siRNA for 48 h were stained with anti-hSpindly and anti–α-tubulin antibodies (green), and CREST serum (red) and DAPI (blue). (C) Cells were treated with GL2, hSpindly-1, or hSpindly-2 siRNAs for 48 h (and treated with or without MG132 for 2 h) and the percentage of mitotic cells with all chromosomes aligned was determined. Bar graphs represent the results of three independent experiments (>100 cells each, only prometaphase and metaphase cells were counted) and error bars indicate SD. (D) Representative stills from videos of H2B-GFP expressing HeLa S3 cells treated with GL2, hSpindly-2, and ZW10 siRNAs for 48 h before filming. Time is shown in h:min. t = 0 was defined as the time point one frame before chromosome condensation became evident. (E) Box-and-whisker plot showing the time cells spent in mitosis from nuclear envelope breakdown (NEBD) to anaphase after treatment with GL2 (control), hSpindly-1 and hSpindly-2 siRNA (3 experiments, >80 cells per experiment, P < 0.0001), or ZW10 siRNAs (2 experiments, >80 cells per experiment, P < 0.0001). Bars, 10 µm.