Supplementary Figure 12: Illustration of the image analysis.

a–h, Successive steps of image analysis to obtain full width at half-maximum (FWHM) of F-actin cortex in early mitosis. Deconvolved image of the LifeAct signal from L1210 FUCCI cells expressing LifeAct–RFP F-actin probe (a). The image after application of the median filter (b). Binary converted image (c). Automatic detection of cell boundaries (red circle) (d). 100 radial paths (yellow lines) on which the LifeAct signal was quantified (e). Overlay of all radial path signals (yellow) as a function of distance from the cell center (f). The median of the radial paths (black line) was used to determine baseline within the cell, maximum signal at the actin cortex and, consequently, the FWHM. g,h, Nuclear envelope breakdown (NEBD) was used for aligning different cells to the same point in the cell cycle. NEBD was detected by the abrupt spread of the green Geminin fluorescence of the FUCCI from a restricted nuclear localization (g) to the whole cytoplasm (h). i–l, Successive steps of image analysis to detect equatorial and polar regions of the F-actin cortex in late mitosis. Steps in order (top to bottom) and from different time points (left to right): deconvolved image of an L1210 FUCCI cell expressing LifeAct–RFP F-actin probe (i). Detection of one or two cells in the image (red circles) (j). Assignment of the polar regions (white areas) (k). Assignment of the equatorial regions (white areas) (l). See Methods for additional details.