Short form of α9 structure. (A) Schematic representation of α9 integrin and SFα9 protein. The β-propeller, thigh, genu, calf-1, calf-2, transmembrane (TM), and cytoplasmic (CYT) domains are indicated. The SFα9 protein is identical to α9 integrin up to thigh domain and contains 19 novel amino acids. (B) Schematic representation of α9 integrin and SFα9 genome and cDNA. The cDNA sequence coded 19 novel amino acids in SFα9 are derived from exon 15b. (C) The cDNA sequence of SFα9 derived from exon 15b. The border between exon15a and 15b has a GT splice donor site. 3' end has poly(A)⁺ tail. The polyadenylation cleavage site (AATAAA) is indicated (arrowhead).

Expression and adhesion of SFα9. (A) SFα9 is not secreted as a soluble protein. Western blot analysis of full-length α9 integrin subunit and SFα9 in cell lysates and culture supernatant from MEF cells transduced with retroviruses encoding the cDNAs of α9 integrin subunit, SFα9, Myc-tagged, FLAG-tagged SFα9, or control. Cell culture supernatants were concentrated 10-fold using centricon tubes (Millipore). Anti-β-actin antibody or anti-osteopontin antibody was used as loading control for cell lysate or culture supernatant, respectively. (B, C) SFα9 is expressed on cell surface. Flow cytometry demonstrating cell surface expression of α9 integrin subunit or SFα9. Shaded peaks represent unstained cells, and open peaks represent cells stained with anti-α9 integrin Y9A2 (B). Cell surface proteins were labeled with thiol-cleavable biotin, captured with streptavidin agarose, then eluted by reduction. Eluted proteins were then analyzed by western blotting(C). (D) SFα9 does not bind to TNfn3RAA (tenascin-C). The binding of MEF cells expressing α9 integrin subunit or SFα9 to the indicated concentrations of TNfn3RAA and fibronectin. Asterisk represents a statistical significant difference (*P < 0.05; **P < 0.005) as compared with adhesion of Mock/MEF cells. Data are presented as means ± SEM of 3 independent experiments.

Endogenously expressed SFα9 promotes α9-dependent cell adhesion. (A)A9A1 is useful antibody for SFα9 immunoprecipitation. Lysates from SFα9-FLAG/α9/SW480 cells were immunoprecipitated by anti-α9 integrin Y9A2, A9A1, or B9A1, precipitated proteins were then separated by SDS-PAGE, and blotted with anti-FLAG antibody. Anti-FLAG antibody for immunoprecipitation was used as positive control. (B) Endogenous expression of SFα9 in RD cells. Lysates from SFα9-FLAG/α9/SW480 cells or various tumor cell lines were immunoprecipitated by anti-α9 integrin A9A1, precipitated proteins were separated by SDS-PAGE, and blotted with anti-α9 antibody (R&D). (C) Flow cytometric evaluation of GFP expression in RD cells transduced with lentiviruses encoding the SFα9 shRNA at 4 days after transduction. (D) Full-length α9 integrin subunit and SFα9 mRNA levels in SFα9 shRNA-transduced RD cells at 4 days after transduction was quantified using real-time PCR and expressed as percent basal levels (normalized to RD cells transduced with pSicoR vector). Asterisk represents a statistical significant difference (*P < 0.05; **P < 0.005) as compared with expression of SFα9 in RD cells transduced with pSicoR vector. (E) Full-length α9 integrin subunit and SFα9 protein in SFα9 shRNA-transduced RD cells at 4 days after transduction was detected by immunoprecipitation, as described in (B). (F) Cell adhesion of SFα9 shRNA-transduced RD cells at 4 days after transduction to TNfn3RAA or plasma fibronectin. Asterisk represents a statistical significant difference (*P < 0.05; **P < 0.005) as compared with adhesion of RD cells transduced with pSicoR vector. Data are presented as means ± SEM of 3 independent experiments.

SFα9 binds to neither full-length α9 integrin subunit nor β1 integrin subunit. Lysates from CHO cells co-expressing full-length α9 integrin subunit and FLAG-tagged SFα9 or control were immunoprecipitated by anti-FLAG antibody or anti-α9 integrin (NC7) antibody. The precipitated proteins were then separated by SDS-PAGE, and blotted with anti-FLAG, NC7, or anti-β1 integrin antibodies. The whole cell lysates (WCL) were immunoblotted with anti-FLAG, NC7, or anti-β1 integrin antibodies.

Expression of full-length α9 integrin subunit or SFα9 in various tumor cells. RT-PCR analysis (A) or real-time PCR analysis (B) of full-length α9 integrin or SFα9 expresion in various tumor cells. (C) Absolute copy number of full-length α9 or SFα9 mRNA. (D) Surface expression of α9 on various tumor cells. Flow cytometry demonstrating cell surface expression of α9 integrin subunit. Shaded peaks represent unstained cells, and open peaks represent cells stained with anti-α9 integrin Y9A2. (E) Cell binding of various tumor cells to TNfn3RAA (20µg/ml). Data are presented as means ± SEM of 3 independent experiments.

SFα9 promotes α9-dependent cell adhesion. (A) Flow cytometric evaluation of cell surface expression of α9 integrin subunit on MEF cells co-transduced with α9 integrin subunit and two different-tagged or non-tagged SFα9. (B) Western blot analysis of full-length α9 integrin subunit and SFα9 in lysates from MEF cells co-transduced with full-length α9 integrin subunit and two different-tagged or non-tagged SFα9. (C) The binding of MEF cells co-expressing full-length α9 integrin subunit and two different-tagged or non-tagged SFα9 to the indicated concentration of TNfn3RAA and fibronectin. Asterisk represents a statistical significant difference (*P < 0.05; **P < 0.005) as compared with adhesion of Mock/α9/MEF cells. Data are presented as means ± SEM of 4 independent experiments.

Effect of α9 integrin subunit cytoplasmic domain on promoting α9-dependent cell adhesion by SFα9. (A) Flow cytometry of cell surface expression of α9 integrin subunit on α9-, α9α5-, α9α4-expressing CHO cells transfected with FLAG-tagged SFα9. Shaded peaks represent unstained cells, and open peaks represent cells stained with anti-α9 integrin Y9A2. (B) Adhesion of α9-, α9α5-, α9α4-expressing CHO cells transfected with FLAG-tagged SFα9 to TNfn3RAA or plasma fibronectin. Asterisk represents a statistical significant difference (*P < 0.05; **P < 0.005) as compared with adhesion of Mock/α9/CHO, Mock/α9α5/CHO, or Mock/α9α4/CHO cells. Data are presented as means ± SEM of 3 independent experiments.