FIGURE 3.

Measurements of chromosome length and width in CAP-D3^T1403A cells. A, the chromosomal scaffold protein KIF4A was used as a marker to identify and measure the length (axis) of mitotic chromosomes, whereas the width was measured by DNA stained with DAPI. KIF4A staining is shown on the left panels and DAPI on the right panels. For scoring purposes, the largest chromosome (chromosome 1) in each spread was measured for length using KIF4A and width using DAPI for DNA (examples in inset). Scale bar represents 5 μm. B, scatter plot analyzing parental DT40, CAP-D3^WT, and CAP-D3^T1403A chromosomes from asynchronous cells. To calculate the width and length, the longest chromosome from each spread (chromosome 1) was analyzed and plotted. Mutant and wild type cells were treated with Dox for 48 h. Two representative CAP-D3^T1403A clones were used. All cells were fixed with methanol:acetic acid and dropped onto slides before immunostaining with anti-KIF4A. C, merged example of immunofluorescence of CAP-D3^WT and CAP-D3^T1403A spreads stained with KIF4A (red) and DAPI (blue) for DNA and used for scoring in A.