EZH2 is required for Twist-induced stemness. (A) Quantitative reverse transcription polymerase chain reaction expression of EZH2 in human mammary epithelial (HMLE) cells expressing the indicated epithelial-mesenchymal transition (EMT) inducers. (B) EZH2 protein levels assessed by western blot in HMLE Twist cells transduced by vectors expressing two different short hairpin RNAs (shRNAs) targeting EZH2 and a control vector (FF3). Note that ShRNA #2 was more efficient in reducing EZH2 protein levels as indicated by western blot. (C) Histone H3 and H3K27me3 protein levels assessed by western blot indicate that shRNA #2 reduces H3K27me3. (D) Mammospheres were counted in HMLE Twist FF3 cells (control) and shEZH2 cells grown in mammosphere-promoting conditions. Spheres greater in size than 50 micrometers after 10 days were counted and a Student’s t-test was performed. (E) Count of HMLE Twist FF3 (control) and shEZH2 cells after multiple days in standard cell culture conditions. (F) Western blot analysis of H3K27me3 level in HMLE Twist cells before and after treatment with 3-deazaneplanocin A. (G) HMLE Twist cells were treated with 2.5 μM of 3-deazaneplanocin A for 8 days followed by 48 hours without drug. Surviving cells were then subjected to a sphere assay in the absence of drug for 8 days. (H) The morphology of HMLE Twist cells treated with 2.5 μM 3-deazaneplanocin A is depicted after 8 days of treatment. We observed a change toward an epithelial morphology.