Figure 4.

Molecular inhibition of Aurora-A kinase activity in vitro reverses EMT and suppresses self-renewal ability of CD24^–/low breast cancer cells. (a) Venn diagram of the unsupervised cluster analysis of global gene expression between vMCF-7^ΔRaf-1 1GX cells treated with Alisertib or with an shRNA Aurora-A vector showing 90% overlap expression profile. (b) Graph showing FACS analysis of vMCF-7^ΔRaf-1 1GX cells treated with 1 μM Alisertib for 48 and 72 h displaying a CD24⁺ phenotype from three independent experiments (±s.d). (c) Immunoblot analysis of CD24⁺ and CD24^–/low cells showing that treatment with 1 μM Alisertib induced cleaved PARP mainly in the CD24^–/low subpopulation. (d) Immunofluorescence analysis of CD24⁺ and CD24^–/low cells treated with 1 μM Alisertib for 48 h showing activation of apoptosis mainly in CD24^–/low cells. Cleaved PARP was labeled in green and DNA was labeled in blue with Hoechst dye. (e) Graph showing the percentage of CD24⁺ and CD24^–/low cells displaying cleaved PARP before and after treatment with Alisertib. Experiments were performed in triplicate (±s.d.). (f) Immunoblot analysis of CD24^–/low cells treated with Alisertib for 48 and 72 h showing reversion of EMT and restoration of an epithelial CD44^–/CD24⁺ phenotype. (g) Immunofluorescence analysis of CD24^–/low cells treated with1 μM MLN8237 for 48 and 72 h showing reversion of EMT. E-cadherin and p-SMAD5 were labeled in red, β-catenin and vimentin were labeled in green, and DNA was labeled in blue with Hoechst dye. (h) Light microscopy analysis showing inhibition of mammospheres growth derived from CD24^–/low cells after treatment with 0.1 μM Alisertib for 72 h. Upper panels: low magnification; lower panels: high magnification. Graph showing the percentage of cells derived from mammospheres before and after treatment with Alisertib. Experiments were performed in triplicate (±s.d.).