Fig. 3

ATR2 siRNA transfection and immunophenotyping for EC markers. I Concentration selection for siRNA transfection. Three different concentrations (10, 35, and 50 nM) of ATR2 siRNA were used according to the manufacturer’s protocol. Western blot analysis showed inhibition of ATR2 by 10, 35, and 50 nM of ATR2 siRNA. However, 50 nM of ATR2 siRNA showed the highest inhibition among all three different concentrations (A). ATR2 silencing by siRNA transfection with EGM compared with AMSCs with EGM and EGM + scrambled siRNA (negative control) (B). GAPDH was used as a housekeeping gene. II Flow cytometric analysis of PECAM1 (CD31) in four different groups; control group with EGM (A), AMSCs with EGM and MMP-2 siRNA (B), AMSCs with EGM and MMP-14 siRNA (C), and HUVECs as the positive control (D). Cell transfection with 5 μM of ATR2 siRNA for EGM (E), AMSCs with EGM and MMP-2 siRNA (F), and AMSCs with EGM and MMP-14 siRNA (G). Flow cytometry data were analyzed to show the significant differences between the groups (H). III Flow cytometric analysis of VE-cadherin (CD144) in four different groups: control group AMSCs with EGM (A), AMSCs with EGM and MMP-2 siRNA (B), AMSCs with EGM and MMP-14 siRNA (C), and HUVECs as the positive control (D). Cell transfection with 5 μM of ATR2 siRNA for EGM (E), AMSCs with EGM and MMP-2 siRNA (F), and AMSCs with EGM and MMP-14 siRNA (G). Flow cytometry data were analyzed to show the significant differences between the groups (H). **p < 0.01. EBM endothelial cell basal medium, EGM endothelial cell growth medium, MMP matrix metalloproteinase, ATR2 angiotensin receptor R2, HUVEC human umbilical vein endothelial cell