Fig. 6

PRC2-mediated epigenetic regulation governs access to EMT states in basal epithelial variants. A Sorted basal epithelial cells (P3) were transduced with lentiviral CRISPR constructs containing CAS9 and sgRNA targeting EZH2 or non-targeting controls (Cont.). These constructs included an IRES-GFP sequence, which facilitated the selection of cells incorporating the vector using FACS. EZH2 and control cells were expanded in MEGM; at each passage, cells were counted, and a growth curve of the cumulative population doubling over time was plotted. B Same as in A, starting with P8 (variant) basal epithelial cells. Cells were collected at P8 + 3, as indicated by the arrows. C Western blot analysis of lysates from P8 EZH2 KO and control cells using antibodies against Zeb, N-cadherin, EZH2, H3K27me3, total Histone H3, and Actin ( loading control). D P-cadherin, N-cadherin, Slug, Snail, Twist, and Zeb were analyzed by qPCR using P8 + 3 EZH2 KO and control cells. E Sorted basal epithelial cells (P3) were continuously treated with 10 μM GSK126, an inhibitor of EZH2, or DMSO (vehicle control) as cells were expanded in MEGM; at each passage, cells were counted, and a growth curve of the cumulative population doubling over time was plotted. F Same as in E, starting with P8 (variant) basal epithelial cells. Cells were collected at P8 + 3, as indicated by the arrows. G P-cadherin, N-cadherin, Slug, Snail, Twist, and Zeb were analyzed by qPCR in P8 + 3 cells treated with GSK126 or DMSO. H In the EMT continuum, cells exist in various states ranging from epithelial to mesenchymal phenotypes, including metastable hybrid transition states. Epigenetic processes involving PRC2 and likely other mechanisms are altered during adaptation to unfavorable cell culture conditions. These alterations influence the transitions between states and contribute to the stabilization of cells within hybrid EMT states