FIGURE 1.

Endorepellin evokes VEGFA down-regulation mediated by α2β1 integrin and SHP-1. A and B, quantification of intracellular VEGFA and FGF2 levels from total HUVEC lysates stimulated with 100 nm endorepellin for 0–10 h as indicated. The values were obtained by immunoblotting and quantified using the Odyssey (LI-COR). C, qPCR of VEGFA and FGF2 transcript levels in response to endorepellin for the specified periods of treatment. Data represent the average fold changes normalized to ACTB (β-actin) ± S.E. (n = 10), ***, p < 0.001. D, in-cell Western assay of HUVECs grown on collagen and treated with the SHP-1 inhibitor NSC87877 (1 μm) for 0–4 h before being fixed. VEGFA levels were determined by immunoblotting with anti-VEGFA followed by IR800-labeled secondary antibody (green) and were normalized to DNA labeled with the far-red fluorescent DNA dye DRAQ5^TM (red). Note that merged images produce a yellow color when VEGFA increases with time upon blocking SHP-1 with NSC87877. E, quantification of VEGFA levels as described in D using the Odyssey Imaging system (LI-COR). F, slot blot of secreted VEGFA from HUVECs treated with endorepellin for 6 h. Where indicated, cells were pretreated for 1 h with Na₃VO₄ (1 μm) or SHP-1 inhibitor (1 μm). G, quantification of secreted VEGFA as described in F normalized on total cell number. H, immunoblot of HUVEC lysates treated with endorepellin (100 nm) and α2β1 blocking antibody (10 μg/ml, mAb 1998Z, Millipore) either alone or in combination. In the latter case, HUVECs were preincubated for 1 h with the blocking antibody before the addition of endorepellin. The lysates were then probed with an anti-VEGFA antibody. I, quantification of VEGFA levels as described in panel H. The values for A and B and D–I represent the mean ± S.E. from four independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.