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Figure 8


Fig. 8. The effect of depletion of topo II{alpha} on the metaphase inter-kinetochore distance. (A) Distances between sister centromeres of human chromosome 11 detected by FISH using D11Z1 DNA as a probe were assessed using Leica Deblur software. Bar, 10 µm. (B) Measurements made of treated cells in parallel with untreated controls (treated/untreated pairs indicated by brackets) are presented as a boxplot showing the third and first quartiles, with the median indicated by a cross in each box. The maximum and minimum values are indicated by the ends of the vertical lines. Each plot is based on ≥40 measurements. In HTETOP cells, topo II{alpha} was depleted by 72 hours exposure to dox. Cells were arrested using MG132 (10 µM for 2 hours 30 minutes), allowing distances under tension to be measured. For the dox-treated, topoII{alpha}-depleted HTETOP cells, data from three independent experiments are presented. In each case, the decrease in the distance across the centromere domain under tension following depletion of topo II{alpha} is significant (P=0.001, 0.0001, 0.01, respectively). To deplete topo IIβ, HTETOP cells were transiently transfected with siRNA (72 hours exposure). Colcemid-treated HTETOP cells served as a control where no spindle or tension existed. The lack of any effect from dox treatment itself was confirmed by analysis of MG132-arrested parental HT1080 cells. Clone J is a derivative of HTETOP that constitutively expresses topo II{alpha} as a fusion with the C-terminus of eGFP (Carpenter and Porter, 2004). (C) Quantification of spindle lengths in HTETOP cells arrested using MG132 (10 µM for 2 hours 30 minutes) following 0 (topo II{alpha}ON) or 72 hours (topo II{alpha}OFF) exposure to dox. Measurements of the pole-to-pole distance, based on {gamma}-tubulin and DAPI staining, were collected from three independent experiments (topo II{alpha}ON, n=154; topo II{alpha}OFF, n=151).





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