Comparison of helicity quantification methods and comparison of spindle shape across molecular perturbations. (A) Schematic diagrams illustrating three methods of quantifying spindle helicity. The optical flow method is calculated from all pixels in an end-on (XZ) view of the spindle, the bundle tracing method is calculated from ∼10 bundles per cell manually traced from an end-on (XZ) view of the spindle, and the bundle angle method is calculated from six bundles in a top-down (XY) view of the spindle. (B) Scatterplots comparing the helicity of the same 18 siCtrl (gray) and 14 siLGN (black) cells analyzed by each of the three quantification methods. Pearson’s correlation coefficients are shown on each plot. (C–E) Violin plots with internal boxplots of spindle length (C), width (D), and aspect ratio (length/width, E) for spindles subjected to molecular perturbations. n.s. not significant, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005, one-way ANOVA with Tukey’s post-hoc test. (F–H) Scatterplots of spindle length (F), width (G), and aspect ratio (H) versus spindle helicity, averaged for each experimental condition. Vertical and horizontal gray lines indicate standard deviation. Helicity is not significantly correlated with spindle length, width, or aspect ratio (two-sided Pearson’s correlation test). Pearson’s correlation coefficients are provided in the lower right of each plot.