Journal: PLoS ONE

Article Title: Adducin Is Involved in Endothelial Barrier Stabilization

doi: 10.1371/journal.pone.0126213

Figure Lengend Snippet: (A) Protein abundance of α- and γ-adducins was evaluated by Western blot. α- tubulin or ß-actin were used to control for equal gel loading. (B) Expression profile of both adducin isoforms in bar graph representing the signal intensity analyzed by densitometry and normalized to the protein level of the respective loading control (Means ± SEM). In comparison to MyEnd cells, HDMEC cells showed significantly higher expression of α- and γ-adducins in total cell lysates (*P<0.05, **P<0.01, paired t-test, 2-tails) (C-F) Dual immunostaining for α-adducin with either AJ or TJ proteins in HDMEC and MyEnd cells revealed colocalization of α-adducin with VE-cadherin (AJ) and claudin-5 (TJ), respectively. The results are representative of three or more independent experiments. Scale bar = 20 μm.

Article Snippet: Human Dermal Microvascular Endothelial Cells (HDMEC), the corresponding maintaining media and the Detach kit for further cell passaging were purchased either from PromoCell (Heidelberg, Germany) or from Lonza (Switzerland).

Techniques: Quantitative Proteomics, Western Blot, Control, Expressing, Comparison, Immunostaining

Journal: PLoS ONE

Article Title: Adducin Is Involved in Endothelial Barrier Stabilization

doi: 10.1371/journal.pone.0126213

Figure Lengend Snippet: Integrity and structural alterations in endothelial cell monolayers, subjected to a Ca 2+ -switch assay, were further evaluated either by TER measurements or by immunofuorescence analysis. (A-B) Time course of mean TER values showed that Ca 2+ -depletion (indicated by first arrow) induced a significant drop in TER which was largely recovered by Ca 2+ -repletion (second arrow) in HDMEC (A) and MyEnd (B) cells. *p ≤ 0.05 difference between control cells and cells depleted of Ca 2+ . Immunofluorescence analysis in HDMEC (C-E) and MyEnd cells (F-H) was performed. After subsequent fixation and permeabilization, cell monolayers were stained for VE-cadherin, α-adducin and α-adducin (pSer481). Additionally, Alexa Fluor 488 phalloidin was used for staining of F-actin. To identify the nuclei, cells were counterstained for DAPI (blue). (C and F) Immunofluorescence in HDMEC (C) and MyEnd cells (F) revealed that under control condition, in parallel to VE-cadherin, α-adducin and α-adducin (pSer481) are markedly localized along cell-cell borders (arrows). (D and J) In both cell lines Ca 2+ -depletion led to irregular (arrows) or absent VE-cadherin staining, associated with reduced localization or complete absence of α-adducin and α-adducin (pSer481) at cell-cell-junctions. This was accompanied with reduced staining of F-actin and induced intercellular gap formation (arrowhead). (E and H) In contrast, repletion of Ca 2+ restored linear VE-cadherin staining as well as accumulation of α-adducin and α-adducin (pSer481) at cell-cell borders (stars). The staining of F-actin was increased as well. For all conditions, no morphological changes in the nuclei were observed. Images are representative of three or more independent experiments. Scale bar = 20 μm.

Article Snippet: Human Dermal Microvascular Endothelial Cells (HDMEC), the corresponding maintaining media and the Detach kit for further cell passaging were purchased either from PromoCell (Heidelberg, Germany) or from Lonza (Switzerland).

Techniques: Control, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Adducin Is Involved in Endothelial Barrier Stabilization

doi: 10.1371/journal.pone.0126213

Figure Lengend Snippet: Barrier integrity of HDMEC control monolayers and monolayers exposed to different inflammatory mediators such as thrombin, LPS and TNFα was evaluated by TER measurements and immunofluorescence analysis. (A) TER in control cells or HDMEC treated with inflammatory agents. Arrows denote the application of mediators. Results are expressed as a mean ± SEM (n = 2). Mediator-induced decrease in TER was accompanied by alterations in the immunostaining pattern not only of the adhesion junctional protein VE-cadherin but also of α-adducin and α-adducin (pSer481). (B-E) HDMEC monolayers exposed to inflammatory mediators or control cells were immunostained for VE-cadherin, α-adducin and α-adducin (pSer481). Staining for F-actin and for nuclei was also accomplished with Alexa Fluor 488 and DAPI, respectively. In comparison to control condition (B) where VE-cadherin was linearly distributed along cell borders and α-adducin as well as α-adducin (pSer481) were present at cell-cell contacts (arrows, B), treatment with LPS (C), thrombin (D) and TNFα (E) led to considerable intercellular gap formation (arrowheads, C-E), paralleled by pronounced increased stress fiber formation and fragmented VE-cadherin distribution. This effect was associated with almost complete loss of α-adducin membrane staining. No differences in the nuclear morphology were observed under these conditions. Scale bar = 20 μm.

Article Snippet: Human Dermal Microvascular Endothelial Cells (HDMEC), the corresponding maintaining media and the Detach kit for further cell passaging were purchased either from PromoCell (Heidelberg, Germany) or from Lonza (Switzerland).

Techniques: Control, Immunofluorescence, Immunostaining, Staining, Comparison, Membrane