Fig. 4

Depletion of EMT transcription factors prevents stasis in basal epithelial cells. A Basal epithelial cells (P1) were transduced with lentiviral CRISPR constructs, containing CAS9 and sgRNA targeting Slug, Snail, Zeb, Twsit, or non-targeting controls (Cont.). These constructs also included an Internal Ribosome Entry Site (IRES)-GFP sequence, facilitating the selection of cells incorporating the vector using FACS. Snail knockout (KO), Slug KO, and control cells were expanded in MEGM, cells were counted at each passage, and a growth curve of the cumulative population doubling over time was plotted. Cells and CM were collected at P2 + 3 (P2 indicates that the transduced cells were at passage 2, and + 3 indicates the number of passages following GFP⁺ selection), as indicated by the arrows. B P-cadherin, N-cadherin, Slug, Snail, Twist and Zeb were analyzed by qPCR using cDNA produced from basal epithelial cells at P2 + 3, selected for integration of Snail KO, Slug KO, or the control vector. C p21 and p16 were analyzed as described in B. D Sandwich ELISA kits were used to determine the concentrations of TGF-β and activin A. An equal number of cells were harvested from basal epithelial cells at P2 + 3 selected for integration of Snail KO, Slug KO, or control vector, and cultured in basal media (supplements excluded) for 24 h. Concentrations were normalized to the final cell number. E Graphic illustration of the key findings presented in Figs. 3 and 4. In control basal epithelial cell cultures in serum-free MEGM, positive feedback between activation of the TGF-β pathway and induction of EMT generates an environment that suppresses proliferation, causing stasis. In basal epithelial cell cultures treated with a TGF-β inhibitor or transduced with lentiviral CRISPR vectors targeting Snail or Slug, the levels of TGF-β pathway activation and EMT induction, as well as the positive feedback loop between them, were diminished, permitting proliferation and avoiding stasis