Supplementary Material for: Cx43 facilitates mesenchymal transition of endothelial cells induced by shear stress

Objectives: This study aimed to determine the function of Cx43 in the endothelial-to-mesenchymal transition (EndMT) process of endothelial cells (ECs) and to explore the potential signaling pathways underlying these functions. Methods: Endothelial cells (ECs) were extracted from rat aorta. ECs was transfected with Cx43 cDNA, Cx43 siRNA and then exposed to 5 or 12 dyne/cm2. Immunofluorescence staining was used to detect the expression of SM22α in ECs, Cx43 and acetylated-α tubulin in ECs. Western Blotting was used to detected the protein expression of α-SMA, CD31, Cx43, H1-caponin, Ift88 and p-smad3 in ECs. Results: The expression of αSMA, SM22α and Cx43 was significantly increased and CD31 was markedly decreased in ECs treated with laminar shear stress at 5 dyn/cm2. The Cx43 cDNA transfection could induce the expression of SM22α or H1-caponin, and attenuate CD31 expression in the ECs. Also, Cx43 overexpression harms cilia formation in ECs exposed to 5dyn/cm2, accompanied with the regulated Ift88 and smad signaling. Conclusions: This study found that laminar shear stress at 5 dyn/cm2 would increase the expression of Cx43, and interfere with primary cilia formation by inhibiting the expression of Ift88 in ECs.