Figure 2
From: Distinct routes to metastasis: plasticity-dependent and plasticity-independent pathways
Design and validation of lineage-tracing reporters of MET. (a) Conceptual design of E-cadCreIIIcI2. In mesenchymal cells, the E-cadherin promoter is inactive and the IIIc exon is included; no Cre is produced. In epithelial cells, Cre is actively transcribed from the E-cadherin promoter and exon IIIc is efficiently skipped, leading to expression of Cre recombinase. E-cadCreIIIcI2 acts on RG, which contains a DsRed ORF and stop codon (red octagons) flanked by Loxp sites (triangles) followed by the EGFP ORF and a stop codon. MET leads to activation of Cre recombinase and a switch from DsRed to EGFP expression. (b) DT and AT3 cells were stably transfected with RG. Cells subsequently transfected with pcDNA6 exclusively expressed DsRed, and cells harboring the Cre ORF activated EGFP expression. When transfected with the E-cadCreIIIcI2 reporter, only epithelial DT cells switched from DsRed to EGFP expression, while AT3 cells maintained DsRed expression. Scale bar=50 μm. (c) Flow-cytometry analysis shows that a small sub-population of AT3 cells undergo MET. (d) RT-qPCR of DT and AT3 cells transfected with RG and E-cadCreIIIcI2 reveals approximately 10-fold higher expression of Cre in epithelial DT cells than in mesenchymal AT3 cells.
