Figure 4.
Figure 4. Cytosolic interaction between MLL5 and PLK1 is required for spindle bipolarity. (A) An increase in PLK1 aggregates after down-regulation of cytosolic MLL5. U2OS cells were transfected with NC- or PLK4-siRNA for 24 h and further transduced with NC- or MLL5-shRNA through lentiviral infection for another 24 h. Cells were then synchronized to metaphase by nocodazole-MG132 treatment and immunostained for PLK1 (red) and centrin-2 (green). Arrowhead indicates PLK1 aggregate. (B) Quantitation of cells with PLK1 aggregates in A. The experiments were repeated three times (n = 50 cells per sample). Error bars represent SEM. 2*, 2 centrosomes; 1*, 1 centrosome; 0*, 0 centrosome. ***, P < 0.001. (C) An increase in multipolar spindles after down-regulation of cytosolic MLL5. U2OS cells were introduced with NC- or PLK4-siRNA for 24 h and NC- or MLL5-shRNA for another 24 h. Cells were then synchronized to metaphase and immunostained for α-tubulin (red) and centrin-2 (green). Insets show high-magnification (2.5×) images of centrioles. (D) Quantitation of cells with monopolar, bipolar, and multipolar spindles in C. The experiments were repeated three times (n = 50 cells per sample). 2*, 2 centrosomes; 1*, 1 centrosome; 0*, 0 centrosome. ***, P < 0.001. (E) Cytosolic interaction between MLL5 and PLK1 during mitosis. Mitotic U2OS cell lysates were ultracentrifuged at 95,000 rpm with a TLA100.3 rotor after low-speed centrifugation (12,000 g). Supernatants before (LSL) and after (HSL) ultracentrifugation were then immunoprecipitated with anti-MLL5 antibody or rabbit IgG and detected by Western blotting with anti-MLL5 and anti-PLK1 antibodies. (F) Cytosolic interaction between FLAG-MLL5 and HA-PLK1 during mitosis. HSL and LSL of 293T cells coexpressing FLAG-MLL5 and HA-PLK1 were immunoprecipitated with anti-FLAG antibody or mouse IgG and detected by Western blotting (WB) with anti-FLAG and anti-HA antibodies. Bars, 10 µm. DNA in A and C was counterstained with DAPI (blue). Results in E and F are representative of at least two experimental repeats.

Cytosolic interaction between MLL5 and PLK1 is required for spindle bipolarity. (A) An increase in PLK1 aggregates after down-regulation of cytosolic MLL5. U2OS cells were transfected with NC- or PLK4-siRNA for 24 h and further transduced with NC- or MLL5-shRNA through lentiviral infection for another 24 h. Cells were then synchronized to metaphase by nocodazole-MG132 treatment and immunostained for PLK1 (red) and centrin-2 (green). Arrowhead indicates PLK1 aggregate. (B) Quantitation of cells with PLK1 aggregates in A. The experiments were repeated three times (n = 50 cells per sample). Error bars represent SEM. 2*, 2 centrosomes; 1*, 1 centrosome; 0*, 0 centrosome. ***, P < 0.001. (C) An increase in multipolar spindles after down-regulation of cytosolic MLL5. U2OS cells were introduced with NC- or PLK4-siRNA for 24 h and NC- or MLL5-shRNA for another 24 h. Cells were then synchronized to metaphase and immunostained for α-tubulin (red) and centrin-2 (green). Insets show high-magnification (2.5×) images of centrioles. (D) Quantitation of cells with monopolar, bipolar, and multipolar spindles in C. The experiments were repeated three times (n = 50 cells per sample). 2*, 2 centrosomes; 1*, 1 centrosome; 0*, 0 centrosome. ***, P < 0.001. (E) Cytosolic interaction between MLL5 and PLK1 during mitosis. Mitotic U2OS cell lysates were ultracentrifuged at 95,000 rpm with a TLA100.3 rotor after low-speed centrifugation (12,000 g). Supernatants before (LSL) and after (HSL) ultracentrifugation were then immunoprecipitated with anti-MLL5 antibody or rabbit IgG and detected by Western blotting with anti-MLL5 and anti-PLK1 antibodies. (F) Cytosolic interaction between FLAG-MLL5 and HA-PLK1 during mitosis. HSL and LSL of 293T cells coexpressing FLAG-MLL5 and HA-PLK1 were immunoprecipitated with anti-FLAG antibody or mouse IgG and detected by Western blotting (WB) with anti-FLAG and anti-HA antibodies. Bars, 10 µm. DNA in A and C was counterstained with DAPI (blue). Results in E and F are representative of at least two experimental repeats.

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