Supplementary Figure 4: Kinetics of APC/C inhibition after chromosome detachment for 11 cells.

HeLa cells expressing H2B-mCherry and securin-mEGFP were imaged by 3D-confocal microscopy from prophase until metaphase. At different time points during metaphase (indicated by black star), the spindle was cut by a pulsed 915 nm laser to detach individual chromosomes. Time = 0 min at the first image acquired immediately after laser microsurgery. Red indicates time points where individual chromosomes were dealigned from the metaphase plate. Dashed lines indicate anaphase, with (red) or without (green) unaligned chromosomes. The gaps in the curve indicate pauses during time-lapse imaging to reduce light exposure. (a) Background-subtracted total fluorescence was normalized to prometaphase as shown in Fig. 4c,d. The first two panels represent the cells shown in Fig. 4. (b) Raw measurements of total fluorescence intensity in mean intensity projections of z-stacks. Gray curves indicate measurements outside cells used for background subtraction.