The activity of Usp16 is enhanced by Plk1 phosphorylation. (A) In vitro deubiquitination assay in the presence of Xenopus Usp16 (xUsp16), xUsp16 + Xenopus Plk1 (Plx1), or xUsp16 + Plx1 + CDK1 where xUsp16 was pretreated with CDK1. Total histones extracted from HeLa cells were used as deubiquitination substrates. Blots of ubH2A (top) and total histone H2A (loading control; bottom) are shown. (B, top) Immunoblot of ubH2A in lysates from asynchronous HeLa cells treated with DMSO or BI2536. (Bottom) Histone H4 was blotted as a loading control. (C) The intensity of immunoblot bands of ubH2A shown in B. The experiment was repeated three times. (D, top) Immunoblot of Plk1 in Plk1 IP complexes precipitated from mitosis-arrested HeLa cells expressing ubiquitin and Myc-tagged WT Usp16, 3E (S330E/S386E/S486E), 3A (S330A/S386A/S486A), or C205S mutant. (Bottom) Immunoblot of Myc-tagged Usp16. (E) Percentage of cells with chromosome misalignment shown in G, determined from three independent experiments with n = 200–250. ***, P < 0.001. (F) Cells with chromosome misalignment caused by Usp16 knockdown were rescued by expression of WT Usp16 and Usp16 3E, but not Usp16 3A and Usp16 C205S. All Usp16s were expressed from siRNA-resistant plasmids. GFP was transfected as a negative control. White arrows point to misaligned chromosomes. (G) Immunostaining of Plk1 in prometaphase HeLa cells with endogenous Usp16 depleted and the expression of RNAi-resistant WT, 3A, and 3E Usp16. Crest was used as a centromere marker. (H) The fluorescence intensity ratios of Plk1 and Crest shown in G, determined from three independent experiments with n = 100–150. **, P < 0.01. (I) Time-lapse microscopy of Usp16 knockdown HeLa cells expressing RFP-H2B and siRNA-resistant GFP-tagged Usp16 WT, GFP-Usp16 3A, or GFP-Usp16 3E. Error bars indicate the SEM. Bars: (F, G, and I) 10 µm; (G, magnified images) 1 µm.