The IκB super-repressor blocks PEDF-induced antiangiogenesis in vivo. HMVECs were infected with adenoviral vector expressing IκB super-repressor (SR) or with control adenoviral vector expressing β-Gal (AdC). Noninfected HMVECs (NI) were used as control. Total cell extracts were collected and analyzed by Western blot for IκB (A). Note decreased endogenous IκB levels on IκB-SR overexpression. (B) HMVECs, noninfected or infected with AdSR or AdC, were incorporated into subcutaneous Matrigel plugs implanted in nude mice. Angiogenesis was induced with VEGF (200 ng/mL) and blocked by 100nM of the PEDF 34-mer peptide (both incorporated in the Matrigel). At the completion of experiment, the plugs were stained for CD31 (red), apoptosis was visualized by in situ TUNEL (green), and the nuclei highlighted with 4,6-diamidino-2-phenylindole (blue). Apoptotic cells appear white because of superimposed blue, red, and green (white arrows). Images were viewed with a Nikon Eclipse TE 2000-U microscope with a 10×/0.45 objective and Fluoromount G imaging medium (Southern Biotech), and captured with a Nikon LH-M100 C-1 camera. Metamorph, Adobe Photoshop CS3 Extended Version 10.01, and Corel Photo-Paint 10 software were used. (C) Digital images were taken and microvessel density evaluated using MetaMorph software. P values are shown. (D) Apoptosis evaluated on digital images with MetaMorph software. P values were determined by analysis of variance.