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Blood, Vol. 112, Issue 7, 2770-2779, October 1, 2008
p120-Catenin regulates leukocyte transmigration through an effect on VE-cadherin phosphorylation
Blood Alcaide et al.
112: 2770
Supplemental materials for: Alcaide et al
Files in this Data Supplement:
- Figure S1. Infection of HUVEC monolayers with p120-GFP Adv does not alter the distribution of junctional proteins or other catenins (JPG, 100 KB)
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HUVEC were infected with high dose of Adv-p120GFP or remained uninfected (Sham), fixed with 10% formalin, permeabilized and stained with the indicated primary mAbs. Cy-3 conjugated secondary Ab was used for detection. A representative field is shown for each condition.
- Figure S2. Overexpression of p120-GFP does not significantly alter the actin cytoskeleton and differentially affected Trans-Electrical Resistance (TER) (JPG, 121 KB)
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(A) HUVEC were infected with Adv-GFP or with high dose of Adv-p120GFP, fixed with 10% formalin, permeabilized and stained with Phalloidin-Alexa Fluor 546. (B) HUVECs were plated for analysis of TER and infected with Adv-GFP (GFP), or Adv-p120GFP. Increasing doses of both viruses were used in the assay in panel B (g stands for GFP and p stands for p120GFP, doses correspond to the amounts used in Fig 3). TER was monitored in replicate wells for 100 h following the virus addition with an electrical cell-substrate impedance sensing system (ECIS, Applied BioPhysics, Troy, NY).
- Figure S3. The length of time required for PMN to transmigrate HUVEC overexpressing p120GFP is protracted (JPG, 44.7 KB)
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HUVEC were infected with AdV-GFP or with high dose of AdV-p120GFP or AdV-VE-cad-GFP, and transmigration was monitored by live cell imaging as described in Methods. Each PMN was followed from the initial contact with the EC monolayer until transmigration was completed and the time needed for TEM was calculated. Values represent the mean ± SD from 5 separate different experiments, and 5–15 PMN were analyzed in each condition and experiment. P values are indicated, comparing each bar with GFP. ** indicates p<0.001.
- Figure S4. Internalization of VE-cad is not significantly increased during leukocyte TEM in HUVEC or MVEC (JPG, 51.9 KB)
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4 hr TNF-α activated HUVEC (A) or MVEC (B) were incubated with NHS-SS-biotin labeled anti–VE-cad mAb (15µg/ml) for 10min at 15°C. Unbound mAb was removed by rinsing the cells with PBS. Flow buffer alone, or containing mononuclear leukocytes, or the leukocyte cell line HL60 that does not transmigrate, were added followed by 10 min incubation under static conditions to allow transmigration to occur. Monolayers were washed to remove non-adherent leukocytes and cell surface biotin label on Hec-1 mAb was stripped by cleavage of the S-S bond with the non-permeant reducing agent MESNA (20°C, 10 mM MESNA in PBS, 30 min). Monolayers were lysed in sample buffer for western blot studies using Streptavidin-HRP to detect biotinylated VE-cad mAb as described in Methods. Lanes 3–7 indicate duplicate samples for internalized VE-cad in the presence or absence of TEM (normalized and quantified below). The other lanes represent all the necessary controls for the experiment (Total amount of biotinylated VE-cad in lanes 1, 2, 8 and 9 and internalized biotinylated VE-cad in lane 10; Lanes 11 and 12 represent HUVEC that were not stained with biotin-S-S–conjugated VE-cad mAb). (B) Internalization of biotinylated VE-cad is shown in MVEC.
- Video 1. VE-cad and p120 co-localize and form a gap during leukocyte TEM in HUVEC (AVI, 1.41 MB)
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Images were taken every 15 seconds.
- Video 2. PMN remain bound to the cell-cell junctions and do not transmigrate through HUVEC monolayers that express high levels of p120GFP (AVI, 5.23 MB)
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Images were taken every 15 seconds.
- Video 3. PMN remain bound to the cell-cell junctions and do not transmigrate through HUVEC monolayers that express high levels of Y658F-GFP mutated VE-cad (AVI, 1.9 MB)
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Images were taken every 15 seconds.
- Video 4. VE-cad gap formation during leukocyte TEM in MVEC (AVI, 2.88 MB)
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Images were taken every 5 seconds.
- Video 5. VE-cad gap formation during leukocyte TEM in MVEC (AVI, 3.1 MB)
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Images were taken every 5 seconds.
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