Analysis of spindle poles and mitotic spindles

In order to quantify the spindle defects in an unbiased manner, we selected only metaphase cells in which a clear chromosome congression was detected at the metaphase plate. This was accomplished by a 3-h MG-132 block. The pole-to-pole distance was assessed by utilizing Image-J to measure the pole-to-pole distance between pericentrin foci. To measure spindle arc, the angle tool was utilized in Image-J. The second point of the angle tool was consistently placed on the central pericentrin focus and the first and second points were placed 3 microns in length parallel to the α-tubulin-stained mitotic spindle. For the analysis of pole splaying, we measured the length of pericentrin foci at the poles in the control and BCCIP knockdown condition. For quantification of spindle pole components, the polygon tool in Image-J was utilized to trace and measure the intensity of indicated markers at the poles.