Supplementary Figure 5: Nuclear position dictates spindle orientation defect.

(a) Measurement of cell dimensions was carried out in cells expressing mRFP-utrophin. Cell length (a) was measured from cortex-to-cortex in a plane parallel to the spindle, while width was measured from cortex-to-cortex perpendicular to the spindle in the plane of the metaphase plate (n = 50 cells/condition). Cell height (b) was measured by taking optical slices through fixed cells (n = 50 cells/condition). (c) Histogram of measured spindle angles following Kif18A KD. (d) Positional data for centrosomes and nucleus within the cell just prior to entry into mitosis (data taken from last timepoint prior to cell rounding). Plotted are distances measured in live cells from the centrosome-to-cell center, centrosome-to-nuclear center and nucleus-to-cell center. Nucleus and cell center were determined by using Fiji to measure the center-of-mass of an outline of each structure. n = 40 for control cells and n = 37 for Kif25 KD cells. (e,f) Data from d was parsed to separate cells with parallel and angled mitotic spindles in control cells (n = 18 cells parallel and n = 7 cells angled) (e) or Kif25 KD cells (n = 21 cells parallel and n = 15 cells angled) (f). Kif25 KD cells show a statistically significant increase in distance from nuclear center to cell center in angled spindles compared to parallel spindles (2 rightmost columns in f, P = 0.0024). Error bars represent mean ± s.e.m.