Figure 5. Akt-1 is required for EZH2-dependent regulation of mitosis and genomic stability.
A. MCF10A-pLVX-EZH2 cells treated with siRNA control or siRNA-Akt-1, siRNA-Akt-2, or siRNA-Akt-3 were subjected to Dox for 3 days to induce EZH2 overexpression. Giemsa-stained metaphase spread analyses show that siRNA-Akt-1 is sufficient to prevent EZH2-induced polyploidy (≥51 chromosomes). In contrast, EZH2 overexpression increased numerical chromosomal alterations in cells treated with siRNA control, siRNA Akt-2, or siRNA Akt-3. Bar graph shows the mean percentage of cells ± SD. B. Immunofluorescence analysis of mitotic MCF10A- pLVX-EZH2 cells treated with siRNA control or siRNA-Akt-1, siRNA-Akt-2, or siRNA-Akt-3 and were then subjected to Dox for 3 days. p-H3 (Ser10) marks mitotic DNA; Aurora A marks the centrosomes, DAPI marks the nuclei. Note that Akt-1 siRNA prevented EZH2-induced mitotic aberrations and the number of cells with extra centrosomes. Akt-2 siRNA caused a 3-fold decrease in the number of mitotic cells independent of EZH2 overexpression (mitotic index for siRNA control/no Dox = 18.3% and for siRNA control/Dox = 27%; compared to mitotic index for siRNA Akt-2/no Dox = 5.6% and for siRNA Akt-2/Dox = 5%). Akt-3 KD was not able to prevent EZH2-induced chromosomal alterations.