Reduced expression of VEGFR-2 and NRP-1 by short-interfering RNAs. (A) Transfection of siRNA duplexes into HUVECs evaluated by fluorescence microscopy. The image shows a cell culture of HUVEC 6 hours after transfection with a fluorescein-conjugated (nonsilencing) control siRNA duplex. The image was collected using an Olympus 1 × 51 microscope (20 ×/0.40 Ph1 lens; Olympus Optical) with a Retiga 1300 digital camera (Qimaging) and IPLab acquisition software (Scanalytics). (B) Specific silencing of VEGFR-2 and NRP-1 by RNAi. Relative mRNA levels of VEGFR-1, VEGFR-2, and NRP-1 (normalized to glyceraldehydes-3-phosphate dehydrogenase [GAPDH] levels) in transfected HUVEC cultures. Black bars represent control siRNA; red bars, VEGFR-2 siRNA; and cream bars, NRP-1 siRNA. Data shown reflect the means ± SD from 3 RNA preparations obtained 48 hours after transfection of siRNA duplexes. (C) Western blot analysis of NRP-1 and actin levels in control siRNA and NRP-1 siRNA-transfected cells. Cell lysates were obtained 48 hours after transfection. (D) Surface levels of VEGFR-2 detected by flow cytometry in control siRNA (open black curve) and VEGFR-2 siRNA-transfected cells (open green curve). Control (IgG1) is represented by the filled red curve. Cells were obtained 48 hours after transfection.