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Blood, Vol. 112, Issue 4, 1120-1128, August 15, 2008
PGE2 induces angiogenesis via MT1-MMP–mediated activation of the TGFβ/Alk5 signaling pathway
Blood Alfranca et al.
112: 1120
Supplemental materials for: Alfranca et al
Files in this Data Supplement:
- Figure S1. HUVEC were transfected with a scrambled oligonucleotide or an siRNA against MT1-MMP (100nM), using oligofectamine (JPG, 61.2 KB)
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After 48h, cells were harvested and either plated onto coverslips for immunofluorescence experiments or lysed for Western blot analysis. (A) Representative micrographs of cells transfected with the scrambled oligonucleotide or theMT1-MMP siRNA, then treated with 1µM PGE2 for 15′ and immunostained with an anti-MT1-MMP antibody. In the case of MT1-MMP siRNA, a weaker staining due to MT1-MMP knockdown can be observed in some cells (asterisks). In such cells, the formation of MT1-MMP clusters upon PGE2 treatment is abolished, in contrast with neighbouring cells with a normal staining strength, where MT1-MMP membrane relocalisation can be clearly observed (arrows, clusters of MT1-MMP). Similar results were obtained with a different MT1-MMP siRNA (not shown). (B) Western blot of whole lysates from scrambled oligonucleotide or MT1-MMP siRNA-transfected cells, where a partial silencing of MT1-MMP protein can be observed in MT1-MMP siRNA-transfected cells.
- Figure 2. HUVEC were plated onto gelatin-coated coverslips, and treated for 5′ or 15′ with 1µM PGE2 (JPG, 41.6 KB)
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Cells were then fixed and stained with an anti–MT1-MMP antibody in the absence (control) or presence of a 20 molar excess of a peptide containing a sequence from the MT1-MMP catalytic domain (20× peptide; described in Gálvez BG. et al, J. Biol. Chem. 276 (40): 37491–37500, 2001). The figure shows representative micrographs of MT1-MMP stained cells. PGE2 induces a clear redistribution of MT1-MMP to membrane clusters in control cells, and this pattern is abolished when specific anti MT1-MMP antibody binding is prevented by competition with a molar excess of the peptide.
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