Fig 2: siRNA-mediated knock-down of MFGE8 gave impetus to EndMT induced by TGF-ß1 in HUVECs. A, Decreased protein level of MFGE8 in TGF-ß1-stimulated HUVECs by Western blot. n = 4 in each group. B, Related mRNA levels of endothelial and mesenchymal markers by qRT-PCR. C, Cellular immunofluorescence of HUVECs for the expression of CD31 (red), a-SMA (green) and nuclei (blue) (n = 6 in each group; original magnification, ×200). D, Levels of EndMT-associated proteins markers assessed by Western blot and the corresponding quantitative analysis. n = 4 in each group. Data are presented as mean ± SEM. *P < .05, **P < .01 and ***P < .001 vs corresponding control (Ctrl or PBS) group or between the 2 indicated groups (one-way ANOVA followed by Student's t test)

Fig 3: rh-MFGE8 reverses EndMT induced by TGF-ß1 in HUVECs. A, mRNA levels of CD31 and ACTA2 by qRT-PCR. n = 4 in each group. B, Cellular immunofluorescence of HUVECs for the expression level of a-SMA (green) (nuclei (blue); n = 6 in each group; original magnification, ×400). C, Protein levels of CD31 and a-SMA by Western blot. n = 4 in each group. Data are presented as mean ± SEM. *P < .05 and **P < .01 between the 2 indicated groups. #P < .05 and ##P < .01 vs corresponding PBS group (one-way ANOVA followed by Student's t test)

Fig 4: MFGE8 regulates EndMT induced by TGF-ß1 through Smad2/3 signalling pathway and influences the expression of EndMT-related transcription factors. A, B, Expression levels of EndMT-associated transcription factors in HUVECs by qRT-PCR (A) and Western blot (B). n = 4 in each group. C, Typical blot results and quantitative analysis of TGF-ßRI and Smad2/3 pathway in different treatment groups in HUVECs. n = 4 in each group. D, Schematic diagram of the hypothesis. Arrows or lines indicate the activation or inhibition relationship between the two molecules. Data are presented as mean ± SEM. *P < .05, **P < .01, ***P < .001 and NS indicate no significance vs corresponding control (Ctrl or PBS) group or between the 2 indicated groups. #P < .05 and ##P < .01 vs corresponding TGF-ß1 group (one-way ANOVA followed by Student's t test)

Fig 5: MFGE8 silence inhibited angiogenesis and promoted cell migration in HUVECs. A, Representative images of angiogenesis assay in HUVECs under different stimulations (n = 4 in each group; original magnification, up: ×100; down: ×200). B, Evaluation of cell migration ability by scratch assay (n = 4 in each group; original magnification, ×100). C, Influence of MFGE8 knock-down on cell migration assessed by Transwell assay (n = 4 in each group; original magnification, ×200). Data are presented as mean ± SEM. *P < .05, **P < .01 and ***P < .001 vs PBS group or between the 2 indicated groups (one-way ANOVA followed by Student's t test)