Difucosylated oligosaccharide Lewis Y is contained within integrin αvβ3 on RL95-2 cells and required for endometrial receptivity  Dongmei Zhang, Ph.D., Jianxin Wei, M.Sc., Jiao Wang, M.Sc., Shuai Liu, Ph.D., Xiaoqi Wang, Ph.D., Qiu Yan, M.D., Ph.D.  Fertility and Sterility  Volume 95, Issue 4, Pages 1446-1451.e1 (March 2011) DOI: 10.1016/j.fertnstert.2010.04.036 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Integrin αvβ3 and LeY are expressed in both human endometrial tissues of secretory-stage and uterine epithelial RL95-2 cells. (A) The gene expression of integrin αv and β3 in endometrial tissues of secretory-stage and RL95-2 cells was analyzed using RT-PCR. The product of the αv subunit (609 bp) or β3 subunit (200 bp) was identified by 1% agarose gel electrophoresis and stained with ethidum bromide. (B) Total protein isolated from the endometrial tissues of secretory-stage or RL95-2 cells was separated by a 10% SDS-PAGE minigel, transferred onto the nitrocellulose membrane. Protein expression was detected using antibodies directed against the integrin αv subunit (top row) or β3 subunit (bottom row). (C) The expression of LeY was analyzed by immunoblotting using an antibody directed against LeY. (D) Localization of integrin αvβ3 and LeY on RL95-2 cells was detected by immunofluoresence staining. RL95-2 cells plated onto glass coverslips were incubated separately with mouse IgG1 (a), mouse IgM (c), or antibodies directed against integrin αvβ3 (b) or LeY (d), and FITC-conjugated IgG or PE-conjugated IgM was used to determine integrin αvβ3 or LeY, respectively. Magnification ×400. ET: endometrial tissue of secretory stage. RL95: cultured RL95-2 cells. Fertility and Sterility 2011 95, 1446-1451.e1DOI: (10.1016/j.fertnstert.2010.04.036) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 SiRNA targeting FUT4 inhibits the expression of FUT4 and LeY but not the expression of integrin αvβ3 and decreases the adhesion of JAR cells to RL95-2 cells. (A) Gene expression of FUT4 (top row), integrin αv (second row), or β3 (third row) was detected by RT-PCR. Beta-actin was used as an internal control. (B) The protein expression level of FUT4 (top row), integrin αv (second row), β3 (third row), or LeY (fourth row) was detected by immunoblotting. Beta-actin was used as an internal control. (C) Expression of integrin αvβ3 and LeY on the cell surface was examined by flow cytometry. Scrambled siRNA–treated control cells or FUT4 siRNA–transfected cells were incubated with antibodies directed against integrin αvβ3 or LeY, followed by incubation with FITC-conjugated IgG or PE-conjugated IgM to detect integrin αvβ3 or LeY, respectively. Results were obtained using the FACScan flow cytometer. (D) RL95-2 cells were allowed to grow to confluent and form the complete monolayer. The monolayer of RL95-2 cells was monitored under the phase-contrast microscope before JAR cells were delivered. The data presented are the representative mean values from three separate experiments. **P<.01, as compared with the scrambled control. Control: RL95-2 cells transfected with scrambled control siRNA; FUT4 siRNA: RL95-2 cells transfected with FUT4 siRNA; Isotype: RL95-2 cells incubated with mouse IgG1 or mouse IgM to serve as an isotype control. Fertility and Sterility 2011 95, 1446-1451.e1DOI: (10.1016/j.fertnstert.2010.04.036) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Expression of LeY oligosaccharide epitope on integrin αvβ3. (A) Integrin αvβ3 was immunoprecipitated from the total protein of RL95-2 cell lysates with an anti-integrin αvβ3 antibody or an isotype control mouse IgG1 antibody as described in the Materials and Methods section. Immunoprecipitated integrin αv (top row) and integrin β3 (second row) were detected by immunoblotting, and LeY oligosaccharide epitope in the immunoprecipitates was examined with anti-LeY antibody (third row). (B) LeY carried by integrin αvβ3 was identified by ELISA. Total protein (0.2, 1, 5, or 10 μg) from RL95-2 cell lysates was incubated with the anti-integrin αvβ3 antibody precoated onto 96-well plates followed by incubation with anti-LeY antibody and HRP-conjugated IgM. The bound anti-LeY antibody was detected at 450 nm by a spectrophotometer. The data presented are the representative mean values of six separate experiments. Fertility and Sterility 2011 95, 1446-1451.e1DOI: (10.1016/j.fertnstert.2010.04.036) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Abolishing the function of LeY and/or integrin αvβ3 prevents the adhesion of JAR cells to the RL95-2 cells and concurrently inhibits integrin αvβ3/FAK phosphorylation. (A) LeY and integrin αvβ3 on the RL95-2 cells were blocked without (control) or with their specific antibodies as described in the Materials and Methods section. The monolayer of RL95-2 cells was monitored under a phase-contrast microscope to assure that antibody treatment does not alter the cell morphology and impair the monolayer of RL95-2 cells before JAR cells were delivered. After 1 hour of incubation, attached JAR cells were calculated as described in the Materials and Methods section. The data presented are the representative of mean values from three separate experiments. *P<.05; **P<.01, as compared with the untreated control. (B) FAK phosphorylation was analyzed by immunoblotting. The expression of FAK or pFAK was detected with an anti-FAK (top row) or anti-pFAK (bottom row) antibody, respectively. Control: RL95-2 cells without any treatment; FUT4 siRNA: RL95-2 cells transfected with FUT4 siRNA; LeY antibody: RL95-2 cells treated with anti-LeY antibody; αvβ3 antibody: RL95-2 cells treated with anti-integrin αvβ3 antibody. Fertility and Sterility 2011 95, 1446-1451.e1DOI: (10.1016/j.fertnstert.2010.04.036) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions