Fig. 2
The CD44s/ZEB1 feedback loop enables the miR-200/ZEB1 circuit to function as a three-way switch. (a) Top panel: The SNAIL-driven miR-200/ZEB1 circuit including the CD44s/ZEB1 feedback loop. Bottom panel: Bifurcation diagram of ZEB1 mRNA levels for the miR-200/ZEB circuit in response to SNAIL. (b) Top panel: The S 1-S 2-driven miR-200/ZEB1 circuit including the CD44s/ZEB1 feedback loop. S 1 represents a transcriptional inhibition signal on miR-200 and S 2 represents a transcriptional activation signal on ZEB1. Bottom panel: The phase diagram (a two-parameter bifurcation) of the miR-200/ZEB1 circuit driven by signals S 1 and S 2. In (a) and (b), the dotted bar-headed arrows represent the alternative splicing of CD44 mRNAs by ESRP1. Different colors in the bifurcation diagrams represent different co-existences of stable states. For example, the blue colored region marks the tristable phase – {E, E/M, M}, where all three phenotypes - E, E/M and M can be the stable states. In (b), the region marked by black dots in phase diagram represents the parameter region of S 1and S 2 for the existence of the hybrid E/M phenotype – existing either alone - {E/M} or in combination with other stable states – {E, E/M}, {M, E/M} and {E, E/M, M}. (c) Top panel: Relative gene expression levels of ZEB1 and ESRP1 in epithelial (n = 11), hybrid E/M (n = 11) and mesenchymal (n = 37) cell lines from NCI-60. Bottom panel: Pearson’s correlations between gene expression of ESRP1 and ZEB1, VIM, CDH1 and OVOL2. ‘*’ represents P value ≤0.05. ‘**’ represents P value <0.005. ‘***’ represents P value <0.0001. (d) Immunofluorescence images showing different expression patterns of EMT markers in NSCLC cell lines. In the first column, blue is for DAPI, red is for ZEB1 and green is for CDH1. In the second column, blue is for DAPI, red is for CDH1 and green is for VIM. (e) mRNA levels of CDH1, VIM, SNAIL and ZEB1 in NSCLC cell lines. (f) Protein levels of CDH1, VIM, SNAIL, ZEB1 and FOXC2 in NSCLC cell lines. In (e) and (f), H820 and H1437 are epithelial cell lines, H1299 and H2030 are mesenchymal cell lines, H1975 is hybrid E/M cell line and H1944 is a mixture of E and M cells