Usp16 regulates the kinetochore localization of Plk1, leading to proper alignment and timely separation of chromosomes. (A) Immunostaining of Plk1 in prometaphase HeLa cells treated with negative control siRNA or siRNA targeting KLHL22, CUL3, Usp16, or both Usp16 and KLHL22. Crest was used as a centromere marker. (B) The fluorescence intensity ratios of Plk1 and Crest shown in A, determined from three independent experiments with n = 100–150. (C) HeLa cells were transfected with control siRNA or siRNA targeting Usp16, and their lysates were blotted (left three lanes). Plk1 was immunoprecipitated with anti-Plk1 antibody or IgG in a mock IP. BubR1 coimmunoprecipitated with Plk1 was blotted (right three lanes). (D) Immunostaining of Plk1 and BubR1 in HeLa cells treated with either control siRNA or siRNA targeting Usp16. (E) Immunostaining of α-tubulin (α-Tub) and Usp16 in HeLa cells treated with control siRNA or siRNA targeting Usp16. The arrow points to misaligned chromosomes. (F) Percentage of cells with chromosome misalignment shown in E, determined from three independent experiments with n = 200–250. (G) Time-lapse microscopy of RFP-H2B–expressing HeLa cells treated with siRNA targeting Usp16 or control siRNA. Arrows point to misaligned or improperly separated chromosomes. (H) Time from nuclear envelope breakdown (NEBD) to metaphase in HeLa cells shown in G, determined from three independent experiments with n = 20–25. Error bars indicate the SEM. ***, P < 0.001. NC, negative control. Bars: (A, D, E, and G) 10 µm; (A, magnified images) 1 µm.