Figure 5

Influence of EphrinB2/EphB4 signaling on angiogenic markers in WJ-MSCs under serum-deprivation. (a) The mRNA expression levels of EphB4 and EphrinB2 were determined by qRT-PCR analysis in control and serum-deprived WJ-MSCs. GAPDH was used as the endogenous control (n = 4). (b) WJ-MSCs were cultured for 48 h under serum-deprivation in the presence of 8 μM of EphB4 inhibitor, NVP-BHG712. Total EphB4 and phosphorylated EphB4 (p-EphB4) expression levels were quantified by Western blotting to validate the action of NVP-BHG712 (n = 3). Representative blot images are shown. Band intensities were first quantified relative to GAPDH, which was used as a loading control. Then, the relative expression of p-EphB4 with respect to total EphB4 was calculated and plotted. (c) mRNA expression level of ADAMTS13, VEGF and PDGF, under serum-deprivation condition in presence of NVP-BHG712 was analysed by qRT-PCR (n = 3). (d) Protein expression pattern was detected by Western blotting (n = 3) for the above mentioned markers, representative blot images are shown. (e) WJ-MSCs were transfected with ADAMTS13 or NC siRNA and exposed to serum-deprivation stress for 48 h. Total EphB4 and phosphorylated EphB4 (p-EphB4) expression levels were quantified by Western blotting, and plotted; representative blot images are shown (n = 3). (f) WJ-MSCs were treated with NVP-BHG712 for 48 h under serum-deprivation and total ERK and phosphorylated ERK (p-ERK) expression levels were evaluated by Western blotting (n = 3). Representative blot images are shown. Band intensities were first quantified relative to GAPDH, which was used as a loading control. Then, the relative expression of p-ERK with respect to total ERK was calculated and plotted. (g) WJ-MSCs were treated with 30 μM of ERK pathway inhibitor, FR180204, for 48 h under serum-deprivation and mRNA expression levels of ADAMTS13 and angiogenic markers, VEGF and PDGF were analysed by qRT-PCR (n = 3). (h) Protein expression pattern for ADAMTS13, VEGF and PDGF under the same treatment was detected by Western blot analysis and representative blot images are shown (n = 3). Serum-deprived condition has been denoted as no-serum (NS), negative control as NC and control as CT in the figure. Each bar represents mean ± SEM (*p < 0.05, **p < 0.01). Statistical comparisons were assessed using one-way ANOVA followed by Bonferroni’s multiple comparison test. Original blots are presented in Supplementary Fig. S7i–m.