Figure 3.

USP9X Restrains APC/C Activity toward SAC-Dependent Substrates

(A) Time-lapse analysis of endogenous Cyclin B-eYFP degradation in U2OS cells treated with control or USP9X siRNAs and arrested in nocodazole, with or without reversine (0.25 μM), as indicated.

(B and C) Time-lapse analysis of cyclin A-Venus (B) or YFP-Nek2A (C) degradation in HeLa cells treated as in (A).

(D) Time-lapse analysis of Venus-Kif18A degradation during an unperturbed mitosis in HeLa cells treated with control or USP9X siRNAs. Cell images below the graph indicate the point in anaphase when quantification started.

(E) Time-lapse analysis of YFP-Mcl-1 degradation in HeLa cells treated as in (A). Scale bar, 10 μM.

(F) Time-lapse analysis of endogenous cyclin B-eYFP degradation in control or USP9X siRNA-treated U2OS cells without a functional SAC. The SAC was abolished by BubR1 depletion and incubation with a high dose of reversine (1 μM).

(G) Time-lapse analysis of cerulean-cyclin B-R42A degradation in U2OS cells, treated as in (F). Doxycycline was added to express cyclin B-R42A for 44 hr prior to imaging.

All graphs in (A)–(G) show the mean values (±SD) from three independent experiments with ten cells quantified for each condition per experiment. NEB, nuclear envelope breakdown.

See also Figure S3.