Spindle misorientation and increased spindle length after hSpindly depletion. (A) HeLa S3 cells were treated with GL2 and hSpindly siRNAs for 48 h and stained with anti-hSpindly, anti–centrin-3 (green), and anti–α-tubulin (red) antibodies, and DNA (blue). Panels on the right show four representative sections along the z-axis (section numbers are indicated); z-stacks were taken every 0.2 µm. (B) Cells were seeded onto fibronectin-coated coverslips, treated with the indicated siRNAs for 48 h, and synchronized by a thymidine block (overnight) and release (9 h). MG132 was added to the cells 1 h before they were stained with anti-pericentrin (red) and anti–α-tubulin antibodies (green), and DAPI (blue). Bottom panels show the X-Z projection of the pericentrin signal. (C) Box-and-whisker plot showing the spindle angles (α) of cells treated as in B, calculated by measuring the pole-to-pole distance (x) and the vertical distance (z) between two poles after taking Z-stacks every 0.4 µm (illustrated in top panel). Only cells with well-separated spindle poles (x > 7 µm) were counted (>50 cells) (GL2 vs. hSpindly/ZW10/Nde1, P < 0.0002; GL2 vs. CenpE, P > 0.05). (D) Bar graph showing the pole-to-pole distances (x) of the cells in B. Error bars show the SD after measuring distances from >50 cells. Bars, 10 µm.