Supplementary Material

Files in this Data Supplement:

• Supplementary Methods and Results - (PDF)

• Supplementary Figure S1 - 945 KB (JPG)
  Dependence of human MAC deposition on the amount of AdCAGCD59 adenovirus used to infect hepa-1c1c7 cells. Cells were infected with the indicated number of vp/cell of the AdCAGCD59 adenovirus. Three days post-infection cells were subjected to 10% NHS treatment for 5 minutes at 37°C and followed by immunocytochemistry for human MAC (red). Cell nuclei are labeled with DAPI. Images are representative of at least three experiments for each different amount of adenovirus.

• Supplementary Figure S2 - 847 KB (JPG)
  Immunocytochemistry for the RPE cell marker RPE65 on primary mouse RPE cells. Following harvesting from the eyes of 6-10 week old C57Bl/6J mice and one week in culture, cells were fixed and subjected to immunocytochemistry in the presence (Primary) or absence (No Primary, Control) of a primary antibody against mouse RPE65 followed by an appropriate Cy3-conjugated secondary antibody. Cell nuclei are labeled with DAPI. Images are representative of at least three experiments with different cell isolations.

• Supplementary Figure S3 - 1.18 MB (JPG)
  Protection of murine RPE from human MAC deposition by adenovirus-mediated delivery of hCD59 and dependence of the level of protection on the extent of hCD59 expression. (A and B) Flatmounts of eyecups from mouse eyes injected subretinally with the indicated adenovirus mixtures (9:1 ratio, total of 3 x 10⁸ vp/eye) and treated with the anti-mouse emmprin antibody followed by 50% NHS for 7.5 minutes at 37°C six days post-injection. Immunohistochemistry for human MAC (red) shows reduced staining at the area of GFP expression of the AdCACD59+ AdCAGGFP - injected eyecup (B) compared to the control injected eyecup (A). Images are representative of at least three independent experiments (n = at least 10 eyecups for each group of injection). (C) Quantification of GFP fluorescence and MAC immunofluorescence intensity at the area of GFP expression of eyecups from eyes injected as indicated. Note the inverse relationship between GFP and MAC fluorescence intensities on the AdCAGCD59+ AdCAGGFP -injected eyecups (more obvious on the 15-minute serum treatment). Indicated times specify length of serum treatment. Each data point indicates GFP (green) or MAC (red) fluorescence intensity from one eyecup. Eyecups are arranged from left to right in the order of increasing GFP fluorescence intensity. Dotted lines represent means. Graphs comprise data from experiments shown in this figure, panels A and B, as well as Fig. 8A-C and data not shown. N.S., not significant.

• Supplementary Figure S4 - 156 KB (JPG)
  Quantification of mouse emmprin immunofluorescence intensity on the RPE. Graph shows emmprin immunofluorescence intensity (arbitrary units) at the area of GFP expression of eyecups injected with indicated adenoviruses or random areas of eyecups from uninjected eyecups. For each group, 12 images (acquired with a 40X objective) from three eyecups were quantified. Graph comprises data from experiments shown in Fig. 9A, B and data not shown. Data are expressed as means ± s.e.m. N.S., not significant.

• Supplementary Figure S5 - 980 KB (JPG)
  Human MAC deposition on murine cornea and protection of corneal endothelium by ex vivo adenovirus-mediated delivery of hCD59. (A) MAC deposition (red) on 14 mm-thick cross-sections of corneas treated as indicated. (B) hCD59 expression (red) on the endothelium of corneas three days post-ex vivo-infection with 1.5x10⁹ vp of the indicated adenovirus. (C) MAC immunostaining (red) on endothelium of corneas pre-infected with the indicated adenovirus as in B and subjected to the MAC deposition assay as in A, top panel. Graph shows quantification of MAC immunofluorescence intensity (arbitrary units) on the corneal endothelium of twelve sections from four corneas in each group. Graph comprises data from experiments shown in A and C. (D) Emmprin immunostaining (red) on endothelium of corneas subjected to immunohistochemistry three days post-infection with the indicated adenovirus as in B. Graph shows quantification of emmprin immunofluorescence intensity on the corneal endothelium of twelve sections from four corneas in each group. Graph comprises data from experiments shown in D and data not shown. Cell nuclei on all corneal sections were labeled with DAPI. All images are representative of sections obtained from four corneas for each group of infection or treatment. ***p < 0.0001, N.S., not significant.