Fig. 1: Identifying canonical EMT states in lung cancer cells through TGFβ time-course analysis.

a Immunoblot of EMT markers in the presence or absence of TGFβ in three NSCLC cell lines (2 week treatment in HCC827 and H3255 cells, 1 week treatment in A549 cells). b Representative confocal images of HCC827 cells stained for E-Cadherin (red) and Vimentin (green) in the absence (a, b) or presence of TGFβ (c, d) for 10 days. Magnification ×40, scale bar 20 μm. White dotted arrows indicate cells that show loss of E-Cadherin and gain of Vimentin expression in untreated conditions. White solid arrows show enlarged cells on the periphery of epithelial islands that have acquired a pEMT phenotype (E-Cadherin⁺/Vimentin⁺); blue and red solid arrows indicate either enlarged and elongated or small and spindle-shaped cells that have acquired mesenchymal phenotypes (E-Cadherin⁻/Vimentin⁺). c Representative images of HCC827 cells treated with TGFβ (5 ng/mL) for 2, 6, and 10 days under continuous re-seeding conditions (see also Supplementary Figs. 1 and 2 and Methods). Magnification ×10, scale bar 200 μm. Below each image are shown respective E-Cadherin/Vimentin and CD44/CD24 flow cytometry plots. Arrows indicate changes in marker expression during EMT and numbers 1–4 designate the identified canonical EMT states, respectively: Epithelial (E), pEMT, pEMT/Mesenchymal (M), and M* (characterized by CD44^hi/CD24^lo, CSC-like cells). d Color-coded gated EMT canonical states depicted on E-Cadherin/Vimentin flow cytometry plots per experimental condition (see also Supplementary Fig. 1). e EMT state dynamics during TGFβ time course. Color-coded EMT states were calculated and depicted as % of total number of cells at each experimental time-point. f Heatmap summary of EMT marker fold (arsinh) changes relative to control condition when analyzed in all cells (left) and relative to the E state when analyzed in the four canonical EMT states individually at the 10-day TGFβ time-point (right). Source Data are provided as a Source Data file.