ATVB/2003/006486

LEGENDS TO ONLINE FIGURES

Figure I. PCNA expression in neointima vessels.

Sections from early (A and B) or advanced (C and D) lesions were double immunostained with an CD105 (red) (A and C) and with an anti-PCNA antibody (green) (B and D). As secondary antibodies RITC- and FITC-conjugated anti-IgG respectively were used. Original magnification 200 X and 250 X for A and B or C and D respectively. A non immune mouse or rabbit IgG was used as negative control in place of primary antibody (data not shown). (E and F) PCNA localizes in the nucleus of EC lining neovessels. A section from early lesion were immunostained with an anti-PCNA antibody (red) and with DAPI, a specific and quantitative DNA stain (blue). For PCNA staining RITC-conjugated anti-IgG was used as secondary antibody. The nuclear localization of PCNA was confirmed by confocal microscopy as described in Methods Sections.

Figure II VEGF expression.

Hematoxylin/eosin-stained sections from early (A) and advanced lesions (D). Immunofluorescence microscopy of early lesion (B) or advanced lesion (E). The sections were immunostained with an anti-VEGF antibody. As secondary antibody FITC-conjugated anti-IgG was used. As negative control non immune rabbit IgG was used in place of primary antibody (C and F). Original magnification 400X.

Figure III Angio1, but not Angio-2, mediates Erk1-Erk2-MAPK and Akt activation.

Total cell lysates from Angio1- or Angio2-treated EC, were subjected to SDS-PAGE. Proteins were transferred to nitrocellulose filters and the filters were IB with an anti-phospho-Erk1-Erk2-MAPK antibody (A) or with an anti-phospho-Akt antiserum (B) and reprobed with an anti-Erk1-Erk2-MAPK or with an anti-Akt antiserum, respectively. As positive control (+), IL-3-stimulated EC were used. (C) High concentration of Angio2 triggers Akt, but not STAT5 activation Cell lysates from untreated (-) or Angio2-treated (800 ng/ml) EC were subjected to SDS-PAGE and processed as above. The filters were IB with an anti-phospho-Akt antiserum (upper panel) or the anti-phospho-STAT5 antiserum (lower panel) and reprobed with an anti-Akt antiserum and with the anti-STAT5 antiserum respectively. (+) Positive control. Similar results were obtained in four individual experiments

Figure IV Localization of activated STAT5 and p21waf in advanced lesions.

(A) Expression of activated STAT5. Hematoxylin/eosin-stained sections from intimal thickening (a); fatty streak (b); fibromuscular (c); and eroded (d) plaques, were immuno-stained with a FITC-conjugated anti-CD105 and with an anti-phospho-STAT5 antibody. A RITC-conjugated anti-rabbit IgG was used as secondary antibody to recognize the activated STAT5. (B) p21waf expression. Hematoxylin/eosin-stained sections from intimal thickening (e); fatty streak (f); fibromuscular (g) and eroded (h) plaques were immunostained with a FITC-conjugated anti-CD105 antibody and with an anti-p21waf antibody. A RITC-conjugated anti-rabbit IgG was used as secondary antibody to recognize p21waf. Note the positive immunostaining for CD105 but not for the phospho-STAT5 or for the p21waf (green only) (b and f) in fatty streak sections. A double positive immunostaining (merged) for the CD105 and the phospho-STAT5 or the p21waf is present in fibromuscular and eroded plaques (c and d; g and h respectively). The inset of each panel corresponds to the magnification of the delimited area (b, c, d and f, g, h) As negative control non immune rabbit or mouse IgG were used in place of primary antibody (not shown). (C) Phospho-STAT5 and p21waf expression. Intima from non atherosclerotic specimens and neointima from early and advanced atherosclerotic specimens as indicated were homogenized in lysis buffer and analyzed by Western blot. The filters were IB with an anti-phospho-STAT5 antibody, reprobed with an anti-STAT5 or with the anti-p21waf antiserum as indicated. Anti--actin IB shows the equal amount of protein loaded. IL-3 stimulated EC and serum starved EC were used as positive control for phospho-STAT5 and p21waf respectivelly (D) p21SIE2 complex formation. (left panel) Nuclear extracts were prepared from neointima of advanced lesions (lanes 1 to 3 ) and from intima of normal vessel (lane 4). p21SIE2 oligonucleotides were used. ns: non specific. The p21SIE2 complexes contain STAT5 (right panel). Nuclear extracts from neointima of advanced lesion (lanes 1 to 3) were incubated with an anti-STAT5 antibody. The DNA-protein complexes were resolved by non-denaturing PAGE. Seven samples were analyzed with similar results.