Fig. 3

Endoglin regulates FSS-induced, ALK1-mediated BMP signaling depending on serum concentration. A, B Immunoblots using antibodies specific against pSMAD1/5 and Endoglin in cells exposed to 30 min of FSS in presence of A 0.2% or B 20% FCS after Endoglin knock-down or scrambled siRNA controls (upper panels). Densiometric quantifications of SMAD1/5 phosphorylation relative to GAPDH expression (lower panels). Data is presented as mean ± SD from 3 independent experiments. Statistical significance against HSS control was calculated using 2-way ANOVA and Šídák’s post hoc test. Statistical significance in between groups (HSS vs. LSS) was calculated using 2-way ANOVA and Šídák’s post hoc test, ns means p >0.05. C Immunoblot using antibodies specific against pSMAD1/5 and Endoglin in cells exposed to 24 h of FSS after Endoglin knock-down or scrambled siRNA controls in the absence/presence of 50 ng/mL ALK1-Fc (left panel). Densiometric quantification of SMAD1/5 phosphorylation relative to GAPDH expression (right panel). Data is presented as mean ± SD from 3–5 independent experiments. D Quantitative PCR showing gene expression of selected markers after 24-h FSS application in Endoglin knock-down or scrambled siRNA control cells in the absence/presence of 50 ng/mL ALK1-Fc. Data is presented as mean ± SD from 4 independent experiments. Statistical significance was calculated using 2-way ANOVA and Šídák’s post hoc test, ****p < 0.0001,***p < 0.001, **p < 0.01, *p < 0.05. E Phase-contrast and immunofluorescence images of leukocyte adhesion assay on HUVECs transfected with siRNA against Endoglin or scrambled control and subjected to HSS for 24h. F Quantification of leukocyte (THP-1) cell adhesion shown in E. Data is shown as percentage of mean scrambled control adhesion. Statistical significance was calculated using Mann-Whitney test. *p<0.05. G Quantitative PCR showing gene expression of SELP after 24-h HSS application in Endoglin knock-down or scrambled siRNA controls cells in the absence/presence of 50 ng/mL ALK1-Fc. Statistical significance against HSS control was calculated using 2-way ANOVA and Šídák’s post hoc test, ****p < 0.0001,***p < 0.001, **p < 0.01, *p < 0.05