Supplementary Figure 5: Negative feedback and SAC silencing is unaffected by high nocodazole or SGO1 depletion.

(a,b) Quantifications of relative kinetochore intensities of indicated antigens from Flp-in HeLa cells (a), LAP-BUBR1^WT or LAP-BUBR1^ΔKARD-expressing cells (b) treated with a high dose of nocodazole (3.3 μM)³⁵. Different mitotic phases where determined by nuclear morphology; Prophase (Pro), early mitosis (EM; defined as dispersed lightly condensed chromatin) and late mitosis (LM; highly condensed chromatin balls typical of nocodazole-arrested cells). Insets display magnifications of the boxed regions. All kinetochore intensities are relative to the maximum signal in each experiment, except in b, which are relative to the prophase signal in LAP-BUBR1^WT cells. The bar graphs show the mean data (±SD) from at least 10 cells for each condition from 1 experiment, which is representative of 2 experiments. (c) Quantification of relative LAP-BUBR1 kinetochore intensities from time-lapse images of LAP-BUBR1^WT cells transfected with mock or SGO1 siRNA arrested in prometaphase with nocodazole and MG132, prior to treatment with reversine (1 μM) and ZM-447439 (2 μM) for the indicated times. At least 15 cells were quantified for each cell line per experiment and quantifications show the mean data (±SD), relative to the 0 min timepoint, from 3 independent experiments. Bars, 5 μm.