Figure 1. Expression and functional activity of RDD fusion proteins in vitro.

(A) Human fibroblasts transfected with different constructs were analyzed for RDD expression by RT-PCR (lanes: 1, weight marker; 2, untransfected fibroblasts; 3, transfected with pVAX-mock; 4, transfected with pVAX-RDD; 5, transfected with pVAX-RFP-RDD; 6, negative control; 7, plasmid pVAX-RDD [positive control 1]; 8, plasmid pVAX-RFP-RDD [positive control 2]. (B) Murine LL/2 cells were transfected with the pVAX-RFP-RDD or the pVAX-RFP plasmid as indicated. Protein expression was detected by fluorescence microscopy. Original magnification, ×400. (C) HUVEC cultures were scratched in a standardized fashion (wounding of the monolayer), thus creating a cell-free lane (width, 0.5 mm) in each culture. Cultures were then incubated with supernatant from untransfected human fibroblasts (circles), from human fibroblasts transfected with the pVAX-mock plasmid (squares), or the pVAX-RDD plasmid (triangles). Migration of cells into the cell-free lane was monitored. Each data point represents the mean of 10 measurements (± SD). Units on the y-axis: μm. ***P < 0.001. (D) Representative HUVEC cultures described in C. The controls shown here are cultures incubated with supernatant from pVAX-mock–transfected fibroblasts, which were similar to the other controls. The margins of the cell layers are indicated by black lines. Original magnification, ×100. (E) Expression of the RDD target proteins, integrins α_Vβ₃ and α₅β₁, on HUVEC as detected by flow cytometry. Strong signals are detected with antibodies directed against CD49e (α₅ integrin subunit), CD51 (α_V integrin subunit), and a combinatorial epitope of CD51 and CD61 (the α_Vβ₃ integrin heterodimer).