Spindle orientation is perturbed in CHICA-, HMMR-, and DYNLL1-depleted cells. (A) HeLa cells were transfected with control, CHICA, HMMR, DYNLL1 siRNA duplexes for 72 h, or CenpE siRNA duplexes for 48 h. The cells were fixed and then stained for tubulin and pericentrin to define the position of the mitotic spindle, and DAPI to detect DNA. A schematic shows the distances x and z measured from the microscope images and used to calculate α, the angle of spindle rotation and w, the pole-to-pole distance. (B) Samples were Western blotted to test for depletion of the respective target proteins. CHICA-C antibody was used for blotting. (C) Plots showing the angle of spindle rotation for cells depleted of the proteins indicated in the figure (n = 30). The Mann-Whitney test was used to calculate P-values for comparison of control and experimental samples. Median spindle angle was significantly different (P < 0.0001) in the DYNLL1-, HMMR-, and CHICA-depleted cells but not the CenpE-depleted cells (P = 0.8187) when compared with the control. (D) The percentage of cells with rotated spindles is plotted in the graph, error bars show the SEM (n = 100 in three independent experiments).