Figure 2. EZH2-mediated induction of abnormal mitosis and Aurora kinase activity.
A and B. Immunofluorescence analysis of mitotic MCF10A- pLVX-EZH2 cells untreated or treated with Dox for 3 days (A), and in CAL51/KD and controls (B). p-H3 marks mitotic DNA; Aurora A marks the centrosomes. Bar graphs show the percentage of cells with > 2 centrosomes ± SD. The number of mitotic cells per 300 cells was 59 (19%) and 78 (26%) for untreated and Dox treated cells, respectively. The number of mitotic cells per 300 cells was 198 (66%) and 78 (26%) for control and EZH2 KD cells, respectively. C. Western blot analysis of MCF10A-pLVX-EZH2 cells untreated or treated with Nocodazole (50 ng/ml for 20 h at 80% confluence) to induce mitotic arrest. EZH2 overexpression induces Aurora A and B levels in asynchronized cultures, and upregulates p Aurora A and B, p Plk1, and the Aurora kinase substrate p H3 Ser10 in Nocodazole treated cells. D. Western blot of CAL51 cells untreated or treated with Nocodazole. EZH2 KD in CAL51 cells show the opposite effects than EZH2 overexpression.