Figure 2.

PSG1-mediated adhesion of the EVTs is dependent on a direct interaction with integrin α5β1. Micrographs of Swan71 cells on wells coated with 30 μg/mL PSG1-Fc for 1.5 h in serum-free media (A, top) or a protein consisting of just the Fc-tag used as control (B, bottom). Images were taken at 40× magnification. (B) HTR8/SVneo and Swan71 cells were seeded in wells coated with PSG1-His or control protein at various concentrations. Cells were incubated for 2 h at 37 °C and the wells were washed to remove non-adherent cells. Cells remaining in the wells were incubated with cell titer aqueous solution and cell adhesion was quantified as described in Materials and Methods. The adhesion of Swan71 cells to wells coated with 60 µg/mL PSG1-His is considered as 100%. (C) HTR8/SVneo and Swan71 cells, pre-incubated with 500 μM RGD peptide or control (ctrl) peptide for 30 min at room temperature were seeded in wells coated with 30 μg/mL PSG1-His or control protein. The adhesion of control peptide-treated cells to PSG1-His is considered as 100%. (D) HTR8/SVneo cells were pre-incubated with the indicated mAbs for 30 min at RT, after which they were seeded in wells coated with 30 μg/mL PSG1-Fc or control protein. (E) HTR8/SVneo cells were pre-incubated with the indicated mAbs for 30 min at RT and seeded on wells coated with 30 μg/mL of native PSG1 (nPSG1) or control protein. (F) Swan71 cells and purified primary EVTs, pre-incubated with the indicated mAbs were seeded on wells coated with 30 μg/mL PSG1-His or control protein and adhesion assays were performed in 21% oxygen and CoCl₂-induced hypoxic-like condition or in 1% oxygen conditions as described in Materials and Methods. The adhesion of control Ab-treated cells to PSG1 is considered as 100% in (D), (E) and (F). (G) CHO-K1 (integrin α5-expressing) or CHO-B2 (integrin α5-deficient) cells were seeded on wells coated with 30 μg/mL PSG1-Fc or control protein. The adhesion of CHO-K1 cells to PSG1-Fc is considered as 100%. (H) nPSG1 or protein control at 20 μg/mL or bovine fibronectin (FN) at 2 μg/mL was coated on wells. After blocking, 1 μg/mL of α5β1 in 1× TBS/1mM MnCl₂ was added and binding of the integrin was detected with biotin-labeled anti-β1 mAb followed by Streptavidin-HRP. I. Wells were coated with PSG1-Fc or protein control (20 μg/mL). After blocking, 1 μg/mL of α5β1 in 1× TBS/1 mM MnCl₂ was added in combination with 0.5 µM RGD or control peptides and binding of the integrin was detected as indicated in (H). The binding of the integrin to the control protein is considered as 1 in (H) and (I). (J) Wells were coated with 20 μg/mL PSG1-Fc or CEACAM9-Fc. After blocking, α5β1 in 1× TBS/1 mM MnCl₂ was added at various concentrations and binding of the integrin was detected as described above. The graph shows values for PSG1 obtained after subtracting the average of the control values. (K). HTR8/SVneo cells were seeded on wells coated with the indicated proteins (20 μg/mL) and adhesion experiment was carried out as described in Figure 2. Cell adhesion to PSG1-His is considered as 100%. Results shown are mean ± S.D. of triplicates from one representative of three independent experiments. p values were obtained by a one-way ANOVA followed by Sidak’s multiple comparison tests for (H), (I) and (K), by a two-way ANOVA followed by Tukey’s multiple comparison tests for (B), (C), (E) and (F), or followed by Sidak’s multiple comparison tests for (G) (**** p < 0.0001).