Exogenous overexpression of MLL5 rescues PLK1 mislocalization and aMTOC formation in MLL5-KD cells. (A) Overexpression of FLAG-MLL5 but not FLAG-MLL5 with S888A mutation rescuing PLK1 mislocalization in MLL5-KD cells. 293T cells were transfected with NC- or MLL5-siRNA for 16 h followed by overexpression of FLAG-MLL5 or its mutant for 8 h. The cells were then synchronized to prometaphase by monastrol treatment and immunostained for FLAG (green) and PLK1 (red). (B) Quantitation of the data presented in A. The experiments were repeated three times (n = 100 cells per sample). Error bars represent SEM. ***, P < 0.001. (C) Quantitation of centrosomal PLK1 signal at prometaphase in A. (a.u., arbitrary unit). 15 cells were captured per sample manually, and mean pixel intensity of PLK1 was computed. ***, P < 0.001. (D) Overexpression of FLAG-MLL5 but not FLAG-MLL5 with S888A mutation rescuing the formation of aMTOC in MLL5-KD cells. 293T cells were transfected with NC- or MLL5-siRNA for 16 h followed by overexpression of FLAG-MLL5 or its mutant for 8 h. After synchronization to metaphase by nocodazole-MG132 treatment, the cells were immunostained for FLAG (green) and pericentrin (red). (E) Quantitation of the data presented in D. The experiments were repeated three times (n = 100 cells per sample). Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. DNA in A and D was counterstained with DAPI. Bars, 10 µm.