Fig. 1
Transient activation of EMT in stasis HMECs precedes variant emergence. A Human breast tissue samples from three donors were used to generate HMEC cultures in MEGM. The first passage after partial trypsinization was considered passage 1 (P1). At each passage, the cells were counted, and the cumulative population doubling was calculated. The resulting growth curves illustrate three distinct phases: the initial logarithmic growth phase (a), the stasis growth plateau (b), and the second logarithmic growth phase initiated by the expansion of variants (c). Protein, RNA, and conditioned media (CM) were collected from passage 2 (P2) HMECs, stasis HMECs (S), and variant HMECs at two passages beyond stasis (S + 2). Crystal violet-stained flasks depict the pattern of variant emergence from the stasis cultures. The graphic to the right illustrates a mixed population of cultured HMECs encountering the stasis barrier; while most cells are eliminated from the cultures, a small population of variants bypass this ‘stress-induced’ barrier and reinitiates growth. B Cell proliferation was measured in HMEC cultures using the Click-iT EdU cell proliferation assay and quantified by flow cytometry using cells collected from P2, S, and S + 2 cultures, which were re-plated overnight and pulsed with 10 µM EdU for one hour. C Gene expression analysis of p21 and p16 was performed on cDNA produced from cells collected from P2, S, and S + 2 cultures by qPCR. Representative images of P2 and S HMECs immunostained with antibodies against p16 and p21, and counterstained with DAPI are shown. D CM collected directly from P2 and S HMEC cultures (incubated with cells for 24 h) was mixed with an equal amount of fresh MEGM and used to culture freshly isolated HMECs (P1). E Sandwich ELISA kits were used to determine concentrations of TGF-β, Activin A, and PAI-1. An equal number of cells was harvested from P2, S, and S + 2 cultures and cultured in basal media (supplements excluded) for 24 h. Concentrations were normalized to the final cell number. F MMP-2 and FN were analyzed by qPCR. G E-cadherin, N-cadherin, P-cadherin, Slug, Snail, Twist, and Zeb were analyzed by qPCR. H Western blot analysis of lysates from P2, S, and S + 2 cells using antibodies against Snail, Zeb, Slug, E-cadherin (E-Cad.), N-cadherin (N-Cad.), Vimentin, and Actin (loading control)