Fig. 1: Overexpression of Sema7A in HUVECs promotes EndMT.

a Sema7A mRNA expression in the Lenti-Con335-hSema7A-GFP-transduced HUVECs (Sema7A) was analyzed by quantitative polymerase chain reaction (qPCR) relative to Lenti-Con335-GFP-transduced control HUVECs (Con335). Data are mean ± SEM, N = 3, ****p < 0.0001. b Sema7A proteins in Sema7A-HUVECs were analyzed by western blotting normalized to GAPDH relative to Con335-HUVECs. Data are mean ± SEM, N = 3, ***p < 0.001. c There was no significant difference in cell proliferation activity between Con335-HUVECs and Sema7A-HUVECs indicated by CCK-8. d Heatmap shows different expression of endothelial cell (EC) related genes (top), mesenchymal cell related genes (middle), and transcription factor (TF) related to EndMT (bottom) for Con335-HUVECs and Sema7A-HUVECs (p < 0.05). e, f CD31⁺ cells in Con335-HUVECs and Sema7A-HUVECs were detected by a flow cytometer. Con335-HUVECs + IgG (green), Con335-HUVECs + CD31 antibody (red), Sema7A-HUVECs + CD31 antibody (purple) were indicated. Data are mean ± SEM, N = 3, ***p < 0.001. g–j CD31, VE-cadherin (VE-cad), α-SMA, and FSP-1 mRNA level were analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, *p < 0.05; **p < 0.01; ***p < 0.001. k–n CD31, VE-cad, α-SMA, and FSP-1 proteins expression were analyzed by western blotting normalized to GAPDH. Data are mean ± SEM, N = 3. *p < 0.05; **p < 0.01; ***p < 0.001. o Con335-HUVECs and Sema7A-HUVECs morphology were observed by microscope. Scale bar = 50 µm. p Cells were immunofluorescently stained for DAPI (blue)/vimentin (red). Scale bar = 50 µm. q, r Con335-HUVECs and Sema7A-HUVECs were cultured and then subjected to wound healing assay. Phase contrast images are from the start of the assay (0 h) and after 24 h. Location of initial scratch margins indicated by dashed green lines. Scale bar = 100 µm. Data are mean ± SEM, N = 3, **p < 0.01. s, t Transwell cell invasion assay were used to detect the migration ability of cells. Representative images of transwell membranes with cells stained by crystal violet. Data are mean ± SEM, N = 3, **p < 0.01. u Col1α1, Col1α2, Col2α1, Col3α1, and FN3A mRNA level were analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, *p < 0.05; **p < 0.01; ***p < 0.001. Collagen (Col), Fibronectin (FN).