Fig. 8. The effect of depletion of topo II 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 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-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 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 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^ON) or 72 hours (topo II^OFF) exposure to dox. Measurements of the pole-to-pole distance, based on -tubulin and DAPI staining, were collected from three independent experiments (topo II^ON, n=154; topo II^OFF, n=151).