siRNA-mediated AKAP12 and AKAP220 knockdown significantly impaired endothelial barrier function.

<p>(A) Barrier function of subconfluent MyEnd cells was monitored by TER measurements following transfection with siRNA specific for AKAP12 and AKAP220. Non-targeting siRNA was used as a control. The results were compared to cells treated with TAT-Ahx-AKAPis inhibitory peptide. AKAP12 and AKAP220 depletion lead to a significant decrease in TER compared to monolayers transfected with n.t. siRNA. Similar, but more prominent, was the effect obtained after TAT-Ahx-AKAPis application. Data were collected from three or more independent experiments (N ≥3, n ≥12). (B) To test the role of specific AKAPs on cAMP-mediated endothelial barrier formation, TER measurements in cells treated with F/R, 48 hours after successful AKAP-depletion were performed; (n≥10). (C) AKAP12 and AKAP220 knockdown was confirmed by Western blot, 48 hours after transfection. ß-actin was used as an internal gel-loading control. (D) Western blot data were analyzed densitometrically. Normalized intensities for AKAP signals are presented as a bar graph. Data were collected from more than three independent experiments (N ≥3). * p≤0.05, **p≤0.01 and ***p≤0.001 indicate statistically significant difference between the examined group and n.t. siRNA; # p≤0.05 shows statistically significant difference between AKAP12 siRNA and AKAP220 siRNA upon treatment with F/R. The difference was significant starting at 1500 min.