Down-regulation of MLL5 causes multipolar spindle formation. (A) Spindle multipolarity in MLL5-KD cells at metaphase. U2OS cells transfected with NC- or MLL5-siRNA were synchronized to metaphase and immunostained for pericentrin (green) and α-tubulin (red). (B) Two centrosomes in control and MLL5-KD cells at prometaphase. U2OS cells transfected with NC- or MLL5-siRNA were synchronized to prometaphase and immunostained for centrin-2 (green) and γ-tubulin (red). Insets show high-magnification (2.5×) images of a pair of centrioles. (C) Multiple centrosomes in MLL5-KD cells at metaphase after microtubule depolymerization. U2OS cells transfected with NC- or MLL5-siRNA were synchronized to metaphase, treated with nocodazole for 1 h to depolymerize microtubules, and immunostained for γ-tubulin (green) and pericentrin (red). (D) Quantitation of the data presented in A–C. The experiments were repeated three times (n = 100 cells per sample). Error bars represent SEM. **, P < 0.01. (E) Extra MTOC formation in MLL5-KD cells expressing GFP–α-tubulin. U2OS cells stably expressing GFP–α-tubulin were transfected with NC- or MLL5-siRNA for 48 h, and images were taken from prophase to metaphase. Frames taken at the indicated time points (h:min) are shown. (F and G) Multiple PCM foci and two pairs of centrioles are present in MLL5-KD cells. U2OS cells transfected with NC- or MLL5-siRNA were synchronized to metaphase and immunostained for γ-tubulin (green) and pericentrin (red) or for centrin-2 (green) and γ-tubulin (red). Inset in G shows high-magnification (2.5×) image of a pair of centrioles. Bars, 10 µm. DNA in A–C, F, and G was counterstained with DAPI (blue).