Journal: The Journal of Biological Chemistry

Article Title: Urokinase-type Plasminogen Activator (uPA) Promotes Angiogenesis by Attenuating Proline-rich Homeodomain Protein (PRH) Transcription Factor Activity and De-repressing Vascular Endothelial Growth Factor (VEGF) Receptor Expression *

doi: 10.1074/jbc.M115.678490

Figure Lengend Snippet: Sprouting in response to VEGF from the aortae of uPA−/− mice transfected with empty, WT-uPA, and ΔK-uPA LVs. A, aortic ring transfected with control pWPXL vector-based LV, which encodes GFP. Aortic rings isolated from uPA−/− mice were incubated with pWPXL-based LV at 100 multiplicity of infection in EBM-2 medium for 24 h. The rings were then embedded in Matrigel and layered with EBM-2 medium containing VEGF. On day 8, sequences of images were taken in various z sections at 3.3-μm intervals to visualize GFP-expressing cells within the vessel, which were then subjected to three-dimensional reconstitution using ImageJ software. A virtual three-dimensional image of the GFP-expressing cells within the aortic ring is shown. Color-coded sequential position of each z-section is indicated on the scale on the left panel. B, RT PCR analysis of the total RNA samples isolated from the empty-, WT-uPA- and ΔK-uPA-LV-transfected aortic rings. C, sprouting from the aortae of uPA−/− mice transfected with the empty-, WT-uPA-, and ΔK-uPA- LVs. Sprouting was stimulated with VEGF (100 ng/ml) in EBM-2 medium. Representative images from five-six experiments under each of the conditions are shown. D, bar graph showing mean ± S.E. area (top) or length (bottom) of sprouts from aortae of WT or uPA−/− mice after stimulation with VEGF. **, p < 0.001 for value uPA LV versus value for KO con LV; *, p < 0.001 for value uPA LV versus value for KO DK-uPA LV; #, p < 0.01 for value con LV versus value for KO DK-uPA LV.

Article Snippet: The fragment was cut with the Mlu1 and Spe1 restriction enzymes and then cloned into the pWPXL vector (Addgene and D. Trono laboratory, EPFL-SV-GHI-LVG, Station 19, CH-1015, Lausanne, Switzerland) and digested with MluI and SpeI to obtain the muPA/pWPXL lentiviral transfer vector, which encodes full-length mouse uPA.

Techniques: Transfection, Plasmid Preparation, Isolation, Incubation, Infection, Expressing, Software, Reverse Transcription Polymerase Chain Reaction