Fig. 5

EMT generates a spectrum of basal epithelial cell states with varying proliferative and self-renewal capacities. A Phase contrast images of P5 basal epithelial cells demonstrating different morphologies. B Dead cells (DAPI⁺) and doublets were removed, and CD44 and CD24 were visualized using flow cytometry. The CD44^hiCD24^lo, CD44^hiCD24^hi, CD44^loCD24^hi, and CD44^loCD24^lo immunophenotypes were quantified. Each bar represents the basal epithelial cells isolated from a single donor. C Same as B, using antibodies against P-cadherin and N-cadherin. Immunophenotypes were gated based on singly stained controls as P⁻N⁻, P⁺N⁻, P⁺N⁺, and P⁻N⁺. D P-cadherin, N-cadherin, Snail, Slug, Twist, and Zeb were analyzed by qPCR using cDNA produced from basal epithelial cells (P5) directly sorted using FACS based on a P⁺N⁻ or P⁺N⁺ immunophenotype. E P⁺N⁻ or P⁺N⁺ basal epithelial cells (P5) were expanded in MEGM and counted at each passage. F P⁺N⁻, P⁺N⁺, and P⁻N⁺ basal epithelial cells (P5) were sorted into 6-well plates, cultured for 14 days in MEGM, and re-analyzed by flow cytometry. The illustration on the right shows the proportion of cells that transitioned between states in three donors: P⁺N⁻ in blue, P⁺N⁺ in purple, and P⁻N⁺ in red. The conversion rates[85], shown next to each straight arrow, represent the proportion of cells that transitioned from their original FACS-enriched state to another over the 14-day period. The proportion of cells remaining in their original state is indicated next to the curled arrow. G P⁺N⁻, P⁺N⁺, and P⁻N⁺ basal epithelial cells (P5) were sorted into 6-well plates at concentrations of 100, 500, 1000, and 2000 cells and cultured in MEGM until variant colonies were observed. Estimates of variant frequency in the sorted populations and p-values were determined by Extreme Limiting Dilution Analysis [86] using the online resource: https://bioinf.wehi.edu.au/software/elda/. H P⁺N⁻, P⁺N⁺, and P⁻N⁺ basal epithelial cells (P5) were sorted into ultra-low-adherence plates containing mammosphere medium [47] (DMEM/F12 supplemented with B27, EGF, FGF2, and heparin). The mammospheres were visualized after 14 days and counted using a phase-contrast microscope. I P⁺N⁻ and P⁻N⁺ cells were sorted from P8 (variant) basal epithelial cell cultures and re-analyzed by flow cytometry after 14 days. The illustration on the right shows the proportion of cells that transitioned between states in three donors: P⁺N⁻ in blue and P⁺N⁺ in purple, as in F. Note that very few P⁻N⁺ cells were observed in variant cultures (see panel C)