Figure 2.

Structural and functional comparison of HDMECs from the SCLS patient and a normal donor. (A) Acquisition of TEER with days in the culture for SCLS HDMECs compared with normal donor HDMECs. Data were compiled from three experiments. (B) Confocal microscopy of claudin-5. Postconfluent HDMEC monolayers organize claudin-5 into a thin peripheral band along cell junctions coincident with TJ formation (left). Treatment with 0.5 ng/ml TNF disrupts the claudin-5 pattern similarly between normal donor and SCLS HDMECs at 6 h (middle), and the recovery of junctional staining is more pronounced in the normal donor HDMECs after 24 h (right). Data are representative of three experiments. Bars, 50 µm. (C) Quantification of confluent claudin-5 junctional staining indicates significantly decreased recovery in the SCLS HDMECs. *, P < 0.05. (D) Electron microscopy of cellular junctions. Normal donor and SCLS patient HDMECs demonstrate similar patterns of cellular overlap (TIG) and numbers of TJs before stimulation with 0.5 ng/ml TNF, after 6 h both cell types display similar disruptions, and after 24 h the normal donor cells have recovered more overlap and TJs. Bars, 500 nm. (E and F) Quantification of electron microscopy reveals significantly decreased overlap in the SCLS cells (E) and significant recovery of TJ in the normal donor but not the SCLS cells (F). *, P < 0.05. Data are representative of two experimental replicates. (G) Stability of p190BRhoGAP. Normal donor and SCLS HDMECs were treated with 10 μM cycloheximide to maintain high levels of p190BRhoGAP. Data are expressed as means ± SDs.