Figure 5

Mesenchymal phenotypes in ECs ^Mes is regulated by synergistic Notch and TGFβ pathways. A) Semi-quantitative qPCR showing the over-expression of notch transcription factors in sorted ECs^Mes. (*** p < 0.001, ** p < 0.01, mean ± SEM, n = 3). B) Western analysis showing stable p-Smad3 expression in ECs^Norm and ECs^Mes compared to total protein. C) Western analysis demonstrating the activation of Smad5 in ECs^Mes compared to ECs^Norm (top lane). Once ECs^Norm were treated with Jag1 and/or TGFβ1, p-Smad5 was up-regulated (top lane). Hes-1 up-regulation in response to TGFβ1 or Jag1 treatments suggests notch pathway involvement (third lane). Simultaneous supplementation of ligands triggered dramatic Hes-1 up-regulation in ECs^Norm implying a synergistic effect. D) Western analysis displaying p-Smad1/5 down-regulation in response to notch and/or TGFβ inhibition. GSI and SB were added to ECs^Norm-MDA-231 co-cultures, then ECs were sorted for analyzing p-Smad1/5 down-regulation comparing to total protein (left panel). Inhibition also down-regulated Hes-1 activation (right panel). Concurrent inhibitions of ECs^Mes dramatically reduced p-Smad1/5 and Hes-1 activation. E) Confocal images demonstrate endothelial and mesenchymal states of ECs co-cultured with MDA-231^Jag1-KD or MDA-231Scrambled. ECs grown with MDA-231^Jag1-KD demonstrated normal endothelial feature (Upper panels). However, they displayed decreased mesenchymal phenotype compared to ECs^Mes (sorted from MdA-231Scrambled) (bottom panel). Scales are 10 μm. F) Semi-quantitative qPCR compared expression of endothelial and mesenchymal markers in ECs^Mes and ECs sorted from MDA-231^Jag1-KD cells. Silencing Jag1 and notch down-regulation in MDA-231^Jag1-KD counteracted the acquisition of mesenchymal phenotype by ECs. G) Wound healing assay to assess the migratory capacity of ECs sorted from MDA-231^Jag1-KD (ECs St. MDA-231^Jag1-KD) and ECMes (sorted from MDA-231^Scrambled) (left panels). After sorting, a 48-hour wound healing assay was initiated under complete starvation to exclude the possibility of cell proliferation. The invasion property of ECs St. MDA-231^Jag1-KD decreased by 1.6-fold compared to ECs^Mes (right panel).