Knockdown of MLL5 leads to aberrant cytosolic aggregation of PLK1. (A and B) PLK1 aggregation in MLL5-KD cells at prometaphase. U2OS cells transfected with NC- or MLL5-siRNA were synchronized to prometaphase and immunostained for PLK1 (green) and pericentrin (red) or PLK1 (green) and CREST (red). Arrowhead indicates PLK1 aggregate. (C) Quantitation of cells with PLK1 aggregates in A. Experiments were repeated three times (n = 100 cells per sample). Error bars represent SEM. **, P = 0.005. (D) Colocalization of PLK1 and pericentrin. Cells transfected with NC- or MLL5-siRNA were synchronized to metaphase and immunostained for PLK1 (green) and pericentrin (red). Arrow indicates colocalization of PLK1 and pericentrin. Arrowhead indicates PLK1 aggregate. (E) Reduced intensity of centrosomal PLK1 in MLL5-KD cells at prometaphase. U2OS cells transfected with NC- or MLL5-siRNA were synchronized to prometaphase and immunostained for PLK1 (green) and γ-tubulin (red). (F) Quantitation of centrosomal PLK1 signal at prometaphase (a.u., arbitrary unit). Thirty cells were captured per sample manually, and mean pixel intensity of PLK1 was computed. ***, P < 0.001. Bars, 10 µm. DNA in A, B, D, and E was counterstained with DAPI (blue).