Supplementary Figure 1: Validation of the mouse Mad2-EGFP-expressing cell line and further analysis of correlative laser microsurgery, time-lapse imaging, and immunofluorescence staining of the spindle.

(a) Prolonged mitosis in presence of 100 ng/ml nocodazole. HeLa cells stably expressing H2B-mCherry and securin-mEGFP were transfected with non-targeting control siRNA and imaged live after 24 h in presence of 100 ng/ml nocodazole. Time = 0:00 h:min at prometaphase onset. (b) Mitotic slippage after depletion of Mad2. Imaging was as in (a), but this cell was transfected with siRNA targeting human Mad2 (siHsMad2), 24 h prior to imaging. Bars 5 μm. (c) Mitotic slippage induced by transfection of siHsMad2 is suppressed in HeLa cells expressing mouse Mad2-EGFP. Imaging was as in (a, b) using cells stably expressing H2B-mCherry and securin-mEGFP, or H2B-mCherry and mouse-Mad2-EGFP. Mitotic slippage within 3 h after mitotic entry was scored based on the condensation status of H2B-mCherry (n = 30 for each condition). (d) The cell shown in Fig. 1e was cut in metaphase with a pulsed 915 nm laser at the area indicated by the white line, and imaged by 3D-confocal live-cell microscopy. (e) The cell shown in (d) was fixed 2:20 min:s after laser microsurgery and stained for α-tubulin. Bars: 10 μm. (f) Quantification of Mad2-EGFP levels on both sister kinetochores of 18 laser-displaced chromosomes with two Mad2 positive kinetochores.