Supplementary Figure 4: Overexpression of Kif25 leads to mitotic delay.

(a) Mitotic timing was measured following double thymidine block showing that Kif25 KD does not effect the time from NEB to anaphase (n = 46 cells for Kif25 KD and n = 35 for control cells), while Kif25 overexpression does (P < 0.0001, n = 50 cells for Kif25 OE). (b) Inter-centromere distance was measured in cells transfected with mRFP-CenpB and imaged every 3 s for 5 min and compared to a Nocodazole control representing the resting inter-centromere length in the absence of MTs. Measured distances for control (n = 72 centromere pairs from 7 cells), Kif25 OE (n = 76 centromere pairs from 6 cells, P < 0.0001), Kif25 KD (n = 78 centromere pairs from 5 cells, P < 0.0001), and Nocodazole-treated cells (n = 77 centromere pairs from 6 cells, P < 0.0001). (c) Quantification of MAD1 persistence time, taken as the time elapsed between the spindle reaching 8 μm in length and the disappearance of the last Mad1 foci. Kif25 OE causes Mad1 to persist for much longer at the kinetochore (n = 16 cells, P = 0.001). (d) Cells were fixed 72 hr after transient transfection of mCh-H2B or EGFP-Kif25 to count the DNA content of interphase cells. Data are for n = 3 experiments, counting 100 cells for each independent experiment. ^∗P = 0.02. Data are mean ± SEM. Statistics compared using unpaired t-test.