Figure 4
The effect of p31 comet overexpression in Eg5-depleted cells. (a) Scheme of p31comet overexpression in Eg5-depleted cells. (b) FACS analysis of p31comet-overexpressing cells with Eg5-depletion. Cells treated with Eg5 siRNA or control siRNA were infected with EGFP or EGFP-p31comet adenovirus and prepared for FACS analysis. A population of >4Â N DNA content in the cells was shown. (c) Western blotting analysis of p31comet-overexpressing cells with Eg5-depletion. The siRNA-treated cell extracts were subject to western blotting with anti-Eg5 antibody to monitor Eg5 depletion, anti-Securin antibody to monitor cell cycle status, anti-GFP antibody for EGFP and EGFP-p31comet, and anti-Mad2 antibody for a loading control. (d) Apoptosis analysis of Eg5-depleted cells. Eg5-depleted cells overexpressing EGFP or EGFP-p31comet were subject to apoptosis analysis with Multicaspase detection kit. A population of apoptotic cells in each treatment was shown. (e, f) Chromosome analysis of p31comet-overexpressing cells with Eg5-depletion. The arrested cells were fixed in methanol/acetone solution and spread onto glass slides. The chromosome spreads were stained with Giemsa dye and observed with microscopy. Typical chromosome morphologies were shown in (e) and categorized into four types. Fifty chromosome spreads were observed and scored.