Figure 3.
Figure 3. CD97 binds to HUVECs through α5β1 and αvβ3. (A) HUVEC attachment to increasing amounts of plate-bound purified CD97 are shown. CD97 splice variants, possessing an Asp(D) to Glu(E) mutation in the RGD motif, also were used in the adhesion assay (top panel). In addition, cells were treated with a competitive inhibitor of RGD (300 μm) to determine the necessity of the RGD sequence in HUVEC interaction with CD97 (bottom panel). ▪ indicates E/1, 2, 5-RGD-FL; □ indicates E/1, 2, 5-RGE-FL; ▴ indicates E/1-5-RGD-FL; ▵ indicates E/1-5-RGE-FL. (B) HUVECs adhere to CD97 with a distinct morphology. Representative cells are shown following their attachment and spreading on coverslips coated with fibronectin or 10 μg/mL E/1-5-RGD-FL. Fixed cells were observed by staining cellular actin with Texas Red phalloidin and focal adhesions with antivinculin antibody followed by an FITC-conjugated secondary antibody. Image acquisition was performed as described elsewhere.30 (C) Adhesion of HUVECs to CD97 involves α5β1 and αvβ3 integrins. HUVEC binding to fibronectin or 10 μg/mL soluble 3EGF(E/1,2,5-RGD-FL) or 5EGF(E/1-5-RGD-FL) CD97 splice variants was determined. To assess the role of integrins on adhesion, cells were treated with function-blocking anti-integrin monoclonal antibodies (20 μg/mL) for 30 minutes before being added to the adhesion assay plate. (D) Deletion mutants of E/1-5-RGD-FL were used in the adhesion assay. Total attached cells are shown in the top panel and nonrounded cells are shown in the lower panel. E/3-5-RGD-374 containing EGF repeats 3, 4, and 5, as well as the RGD motif, stimulated HUVEC binding, whereas a similar construct containing EGF repeats 4 and 5 and the RGD motif did not. EGF-like repeats in the absence of the RGD motif resulted in only rounded cells binding. indicates E/1-5-RGD-FL; ▪ indicates E/3-5-RGD(374); • indicates E/4, 5-RGD(374); × indicates E/1-5; ▵ indicates E/1, 2, 5. Results shown are representative of at least 10 replicates. Data show mean number of cells per ×200 microscopic field. Error bars represent ± standard error of the mean; n = 5.

CD97 binds to HUVECs through α5β1 and αvβ3. (A) HUVEC attachment to increasing amounts of plate-bound purified CD97 are shown. CD97 splice variants, possessing an Asp(D) to Glu(E) mutation in the RGD motif, also were used in the adhesion assay (top panel). In addition, cells were treated with a competitive inhibitor of RGD (300 μm) to determine the necessity of the RGD sequence in HUVEC interaction with CD97 (bottom panel). ▪ indicates E/1, 2, 5-RGD-FL; □ indicates E/1, 2, 5-RGE-FL; ▴ indicates E/1-5-RGD-FL; ▵ indicates E/1-5-RGE-FL. (B) HUVECs adhere to CD97 with a distinct morphology. Representative cells are shown following their attachment and spreading on coverslips coated with fibronectin or 10 μg/mL E/1-5-RGD-FL. Fixed cells were observed by staining cellular actin with Texas Red phalloidin and focal adhesions with antivinculin antibody followed by an FITC-conjugated secondary antibody. Image acquisition was performed as described elsewhere.30  (C) Adhesion of HUVECs to CD97 involves α5β1 and αvβ3 integrins. HUVEC binding to fibronectin or 10 μg/mL soluble 3EGF(E/1,2,5-RGD-FL) or 5EGF(E/1-5-RGD-FL) CD97 splice variants was determined. To assess the role of integrins on adhesion, cells were treated with function-blocking anti-integrin monoclonal antibodies (20 μg/mL) for 30 minutes before being added to the adhesion assay plate. (D) Deletion mutants of E/1-5-RGD-FL were used in the adhesion assay. Total attached cells are shown in the top panel and nonrounded cells are shown in the lower panel. E/3-5-RGD-374 containing EGF repeats 3, 4, and 5, as well as the RGD motif, stimulated HUVEC binding, whereas a similar construct containing EGF repeats 4 and 5 and the RGD motif did not. EGF-like repeats in the absence of the RGD motif resulted in only rounded cells binding. indicates E/1-5-RGD-FL; ▪ indicates E/3-5-RGD(374); • indicates E/4, 5-RGD(374); × indicates E/1-5; ▵ indicates E/1, 2, 5. Results shown are representative of at least 10 replicates. Data show mean number of cells per ×200 microscopic field. Error bars represent ± standard error of the mean; n = 5.

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