Twist1 knockdown fails to disrupt endothelial cell-cell junction integrity in L-HMVE cells under Ang2 treatment <i>in vitro</i>.

<p><b>A</b>) Immunofluorescence micrographs showing cell-cell junction structure by VE-cadherin staining in L-HMVE cells treated with Twist1 siRNA (#1), Ang2, or both in combination (bar, 50 µm). DAPI staining shows the nucleus of each cell. Arrows show the region where cell-cell junctions are disrupted. As a control, cells were treated with siRNA duplex with an irrelevant sequence. <b>B</b>) Graph showing the quantitation of the total discontinuous area in L-HMVE cells treated with Twist1 siRNA (#1), Ang2, or both in combination in at least 10 fields (*, p<0.05). <b>C</b>) Graph showing endothelial permeability in L-HMVE cells treated with Twist1 siRNA (#1), Ang2, or both in combination (*, p<0.05). Permeability (Pa) values are expressed as percentage of control cells. <b>D</b>) Graphs showing Twist1 and Tie2 mRNA levels in L-HMVE cells treated with Twist1 siRNA (#1), Ang2, or both in combination (<i>top</i>, * p<0.01). Immunoblots showing Twist1, Tie2, and GAPDH protein levels in L-HMVE cells treated with Twist1 siRNA (#1), Ang2, or both in combination (bottom). <b>E</b>) Immunoblots showing levels of active RhoA in L-HMVE cells treated with Twist1 siRNA (#1), Ang2, or both in combination (top). Quantitative results (ratio of active RhoA to total RhoA) were normalized to control siRNA treated Ang2-untreated cells (*, p<0.05). All error bars are s.e.m.