Blood, Vol. 115, Issue 25, 5259-5269, June 24, 2010

Endothelial lumen signaling complexes control 3D matrix–specific tubulogenesis through interdependent Cdc42- and MT1-MMP–mediated events
Blood Sacharidou et al. 115: 5259

Supplemental materials for: Sacharidou et al

Files in this Data Supplement:

• Document 1. Supplemental materials and methods (PDF, 111 KB)
  
  
• Table S1. Single siRNA oligo sequences, primers for cloning into the pIEX-5 or pAdTrack-CMV vectors (PDF, 66.8 KB)
  
  
• Figure S1. Temporal analysis of EC tubulogenesis following siRNA suppression of the components of the lumen signaling complex in 3D collagen matrices (JPG, 813 KB) -
  (A) ECs were treated with Luciferase (control), Jam-A, Jam-B, Jam-C, Par3, Par6b, MT1-MMP, Cdc42 and α2 integrin siRNAs and lumen and tube formation was quantitated over 24 hr from time-lapse movies. Data are shown as average EC lumenal area per high powered field (HPF) ±s.d., n=6, p
  
  
  
  
  
• Figure S2. The EC lumen signaling complex selectively controls EC motility in 3D collagen matrices but not on 2D collagen surfaces (JPG, 460 KB) -
  EC cultures were established in 3D collagen matrices (A) or seeded on 2D collagen surfaces (B) after siRNA treatments for indicated components of the lumen signaling complex and control luciferase. Images show EC motility tracings using GFP-nuclear labeled ECs that were either suspended in 3D collagen matrices or on a 2D collagen surface over a 24 hr period. Time-lapse images were analyzed using MetaMorph software to assess EC motility. (C) Quantification of EC distance travelled from the origin within 3D collagen matrices versus 2D collagen surfaces using MetaMorph software as previously described.¹⁶ Data are shown as total distance travelled over a 24h period, ±s.d., n=10, p
  
  
  
  
  
• Figure S3. Deletion of the MT1-MMP cytoplasmic tail increases EC cell surface expression on 2D collagen surfaces or within 3D collagen matrices (JPG, 329 KB) -
  (A) ECs were treated with the indicated adenoviruses and then were seeded on collagen-coated glass coverslips and stained for MT1-MMP cell surface expression after 24 hr. Photographs were taken and then analyzed using Metamorph software to quantitate fluorescence intensity. Bar equals 100 μm. Graph shows the average stain intensities of MT1-MMP cell surface expression per high powered field (HPF), ± s.d., n=3, p
  
  
  
  
  
• Figure S4. EC lumen signaling complexes control lumen and tube formation in 3D collagen matrices and depend on activated Cdc42 (JPG, 655 KB) -
  (A) ECs were suspended in 3D collagen matrices after their overnight transfection with MT1-MMP-FL-wt-S, MT1-MMP-wt, S-GFP-Cdc42 or GFP-Cdc42 adenoviral constructs. Extracts were prepared at 16 hr under control conditions¹⁶ or in the presence of GDP to test for GTP-dependence. Equal amounts of extracts were incubated with S protein-agarose beads. Eluates were analyzed by western blots using antibodies against Jam-B, Jam-C, Par3, Par6b, MT1-MMP, Cdc42 and α2 integrin. (B) ECs were suspended in 3D collagen matrices after their overnight transfection with MT1-MMP-FL-wt-S, MT1-MMP-wt, S-GFP-Cdc42 and GFP-Cdc42 adenoviral constructs. Extracts were prepared at 16h under control conditions¹⁵ or in the presence of GDP to test for GTP-dependence. Equal amounts of extracts were incubated with GST-PAK-PBD beads. Eluates were analyzed by western blot using antibodies against Jam-B, Jam-C, Par3, Par6b, MT1-MMP, Cdc42 and α2 integrin. (C) ECs were suspended in 3D collagen matrices after their overnight transfection with MT1-MMP-FL-wt-S, MT1-MMP-wt, S-GFP-Cdc42 and GFP-Cdc42 adenoviral constructs. Extracts were prepared at 16 hr under control conditions¹⁶ or in the presence of GDP to test for GTP-dependence. Equal amounts of extracts were incubated with Rhotekin Beads. Eluates were analyzed by western blot using antibodies against RhoA, MT1-MMP and Cdc42.
  
  
  
  
  
  
• Figure S5. MT1-MMP and Jam-C co-localize on the EC cell surface during EC lumen formation in 3D collagen matrices (JPG, 191 KB) -
  ECs were seeded within 3D collagen matrices and allowed to undergo tube morphogenesis over a period of 24 hr. At this time, cultures were fixed with 2% paraformaldehyde and processed for immunostaining in the absence of detergent to assess only cell surface expressed antigens. Cultures were stained with both antibodies to MT1-MMP (a rabbit monoclonal antibody) (A) and Jam-C (a mouse monoclonal antibody) (B) and were photographed. Fluorescent secondary antibodies were shown to specifically react with the appropriate species of the primary antibody and did not cross-react with the other species of antibody. The images were overlaid to assess the degree of MT1-MMP and Jam-C co-localization (C). Bar equals 75μm.
  
  
  
  
  
  
• Video 1. ECs were treated with Luciferase control siRNA and were placed in 3D collagen matrices for 24 hr to assess the degree of EC lumen and tube formation using time-lapse videomicroscopy (MOV, 2.98 MB) -
  Frames were collected every 10 minutes and are shown at 16 frames per second.
  
  
• Video 2. ECs were treated with Jam-B and Jam-C siRNAs and were placed in 3D collagen matrices for 24 hr to assess the degree of EC lumen and tube formation using time-lapse videomicroscopy (MOV, 2.5 MB) -
  Frames were collected every 10 minutes and are shown at 16 frames per second.
  
  
• Video 3. ECs were treated with Jam-A siRNA and were placed in 3D collagen matrices for 24 hr to assess the degree of EC lumen and tube formation using time-lapse videomicroscopy (MOV, 3.08 MB) -
  Frames were collected every 10 minutes and are shown at 16 frames per second.