Fig 1: The heterogeneous cell line SUM149PT contains multiple distinct EMT states that can be isolated as single-cell clones.(A) A schematic of the flow cytometry method used to isolate single-cell clones that present as an epithelial (E), three distinct intermediate (EM1, EM2, and EM3), and two mesenchymal (M1 and M2) EMT states. (B) In vitro assessment of clonal migratory and invasive characteristics as measured in a standard transwell assay (n = 3, SD, ****P < 0.0001, and *P < 0.05). Canonical EMT marker expression levels as determined by (C) quantitative RT-PCR (SD, n = 4) or (D) immunoblotting to rank SUM149 clones along the EMT spectrum. (E) Bright-field and (F) immunofluorescent images of EMT clones in vitro stained with vimentin and E-cadherin displaying cell morphology and marker expression and localization, respectively. (G) EMT signature of EMT clones and parental line generated from the ordinal multinomial logistic regression method of gene scoring and (H) distribution of EMT score of the EMT clones among other breast cancer cell lines from the CCLE. (I) Immunofluorescent staining for E-cadherin (red), vimentin (green), and KRT8 (white) of four triple-negative breast cancer lines (two intermediate, HCC38 and Cal-51; one epithelial, HCC1143; and one mesenchymal, MDA-MB-231) from the CCLE displaying heterogeneous phenotypes.