Fig. 2

Basal epithelial cells exhibit enhanced TGF-β signaling and EMT activation compared to luminal epithelial cells. A Human breast tissues from the same three donors utilized in Fig. 1A were further processed into single cells. The cell suspension was stained with conjugated antibodies against CD10, EpCAM, CD49f, CD2, CD3, CD4, CD16, CD64, CD31, and CD45 then DAPI and conjugated streptavidin. Dead cells (DAPI⁺), doublets, and immune/stromal cell types (CD2⁺, CD3⁺, CD4⁺, CD16⁺, CD64⁺, CD31⁺, and/or CD45⁺) were removed. The breast epithelium was separated into luminal (EpCAM⁺) and basal (EpCAM⁻CD49f⁺CD10⁺) enriched populations by FACS and expanded separately in MEGM in 6-well plates. Notably, these luminal and basal populations contain both mature cell type and stem and/or progenitors. Representative wells were immunostained prior to passaging (P0) with the luminal markers cytokeratin 19 (CK19) and mucin 1 (MUC1) or the basal markers p63 and cytokeratin 14 (CK14). B Basal and luminal epithelial cells were further expanded in MEGM, cells were counted at each passage, and the growth curve of the cumulative population doubling over time was plotted. Cells and CM were collected from basal epithelial cells at passages B(P2), B(P5), and B(P7) and from luminal cells at passages L(P2) and L(P3). Crystal violet-stained flasks depict the pattern of variant cell emergence from basal epithelial cell cultures after stasis. C Sandwich ELISA kits were used to determine the TGF-β and Activin A concentrations. An equal number of cells was harvested from the B(P2), B(P5), L(P2), and L(P3) cultures and then cultured in basal media (supplements excluded) for 24 h. Concentrations were normalized to the final cell number. D Representative images of P2 basal and luminal epithelial cells from two donor tissues immunostained with antibodies against pan-cytokeratin and vimentin and then counterstained with DAPI. E E-cadherin, P-cadherin, N-cadherin, Slug, Snail, Twist, and Zeb were analyzed by qPCR using cDNA produced from B(P2), B(P5), L(P2), and L(P3) cells. F Western blot analysis of lysates from B(P2), B(P5), and B(P7) cultures, using antibodies against N-cadherin (N-Cad.), Snail, Zeb, Slug, and Actin (as loading controls). G Same as C, using basal epithelial cells from the B(P3), B(P5), and B(P8) cultures