Figure 7: The target of miR-181c, PDPK1, in endothelial cells regulates the localization of tight junction proteins, N-cadherin and actin.

(a) Co-immunofluorescence of tight junction proteins (Claudin-5, Occludin and ZO-1), N-cadherin (red) and actin filament (green) after the addition of EVs from D3H2LN, BMD2a or BMD2b cells. Scale bar. 20 μm. (b) Western blot analysis of tight junction proteins, N-cadherin, actin and GAPDH. Proteins were from brain endothelial cells treated with PDPK1 siRNA. This experiment was repeated twice. (c) The TEER value was monitored before (day 4) and after (day 5) the transfection of PDPK1 siRNA or negative control. Error bars represent s.d., Student’s t-test, n=3 (*P<0.01). (d) Luciferase activities measured by cotransfecting miR-181c and the PDPK1 luciferase reporters. Error bars represent s.d., Student’s t-test, n=6 (**P<0.01). (e) Western blot analysis of PDPK1, cofilin, phospho-cofilin (P-cofilin) and GAPDH. Proteins were from brain endothelial cells treated with EVs from D3H2LN, BMD2a or BMD2b cells. This experiment was repeated twice. (f) Western blot analysis of PDPK1, phospho-cofilin (P-cofilin), cofilin and GAPDH. Proteins were from brain endothelial cells treated with miR-181c or PDPK1 siRNA. This experiment was repeated twice. Data are representative of at least three independent experiments each (Fig. a,c,d).