Figure 1.
Structure of CD109 cell-surface glycoprotein.
A, Schematic illustration of FLAG-tagged human CD109 (FLAG-hCD109) structure on cytoplasmic membrane. Anti-CD109-C-9 mAb, anti-FLAG mAb and anti-FLAG pAb can detect 180-kDa N-terminal fragments; anti-CD109-11H3 mAb can detect 25-kDa C-terminal fragments. B, Schematic illustration of construction of FLAG-tagged mouse CD109 (FLAG-mCD109) transgene.
Figure 2.
Detection of soluble CD109 in serum and urine of mCD109-TG mice.
A, Expression of CD109 in various tissues of mCD109-TG (TG-206) mice. Blots of anti-β-actin antibody are shown as internal controls. White and gray arrowheads: 160- and 190-kDa bands, respectively. B, Detection of soluble CD109 in serum of mCD109-TG mice. White arrowheads:160-kDa bands. C, Expression of CD109 in bladder, and soluble CD109 in urine, of mCD109-TG mice. White and gray arrowheads:160- and 190-kDa bands, respectively. Sera, bladder tissues and urine of WT and TG siblings of 3 lines of mCD109-TG mice (TG-203, 206, and 213) were analyzed using western blots with the indicated antibodies; blots of anti-β-actin antibody and IgG light chain were used as internal controls.
Figure 3.
Characterization of HEK293-FLAG-hCD109 and -VC cell lines.
A, Expression of CD109 in total cell lysates and detection of soluble CD109 in culture media of HEK293-FLAG-hCD109 and -VC cell line. Total cell lysates and culture media were subjected to western blotting using the indicated antibodies; blots of anti-β-actin antibody shown as internal controls. B, Cell proliferation analysis of HEK293-FLAG-hCD109 and -VC cell lines. Absorbance values at day 1 are defined as 1.0. NS: not significant, using the Tukey–Kramer's HSD test.
Figure 4.
Characterization of tumors developed in xenografted mice.
A, Schematic illustration of the schedule of xenografted mice experiments using HEK293-FLAG-hCD109 and -VC cell lines. Blood was collected every 7 days after the xenograft until tumor resection, and 17, 48, 72 and 168 h after tumor resection. B, Gross appearance of xenografted tumors in mice. C, Growth curves of xenografted tumors. Seven xenografted tumors of HEK293-FLAG-hCD109 and seven xenografted tumors of HEK293-VC were analyzed for tumor volume, as indicated in Materials and Methods. NS: not significant, using Tukey–Kramer's HSD test. D, Histopathological appearance of xenografted tumors. Sections of xenografted tumors resected at Day 42 were subjected to H&E staining, and immunohistochemical staining with the indicated antibodies. Scale bar: 200 µm. E, Expression of CD109 in xenografted tumors. Total cell lysates from three of each xenografted tumor groups of HEK293-FLAG-hCD109 and -VC cell lines were analyzed for CD109 expression by western blotting. Dotted-white, black and gray arrowheads: 25-, 180- and 190-kDa bands, respectively. Blots of anti-β-actin antibody shown as internal control.
Figure 5.
Proportional increase of soluble CD109 in serum with tumor volume in HEK293-FLAG-hCD109 xenografted mice.
A, Detection of soluble CD109 in serum of HEK293-FLAG-hCD109 xenografted mice. Sera of mCD109-TG (TG-206) mice and xenografted mice were analyzed for CD109 by western blotting with the indicated antibodies. Molecular weight of mCD109: ∼160 kDa (white arrowheads); that of FLAG-hCD109: ∼180 kDa (black arrowheads); western blot for IgG light chain is indicated as an internal control. B, Analysis over time for soluble CD109 in serum of xenografted mice. Serum samples were analyzed for CD109 by western blotting with anti-CD109-C-9 mAb (upper panel). They were also analyzed for tumor-secreted FLAG-hCD109 by immunoprecipitation with Anti-FLAG M2 Affinity Gel, followed by western blotting with anti-CD109-C-9 mAb (middle panel); western blot for IgG light chain is indicated as an internal control. C, CD109 concentration in sera of xenografted mice (HEK293-FLAG-hCD109, N = 7; -VC, N = 7). Tumor-secreted FLAG-hCD109 in sera was assessed by CD109 ELISA, which recognize human CD109, but not mouse CD109. *P<0.01, **P<0.0001, using Tukey–Kramer's HSD test. D, Relationship between concentration of tumor-secreted FLAG-hCD109 in sera with tumor volumes in xenografted mice. Results in C and Fig. 4C were combined and graphically summarized.
Figure 6.
Rapid decrease of tumor-secreted FLAG-hCD109 in serum of HEK293-FLAG-hCD109 xenografted mice after tumor resection.
A, Analysis over time of tumor-secreted FLAG-hCD109 in sera of HEK293-FLAG-hCD109 xenografted mice after tumor resection. Serum samples were analyzed for CD109 by western blotting with anti-CD109-C-9 mAb (upper panel). They were also analyzed for tumor-secreted FLAG-hCD109 by immunoprecipitation with Anti-FLAG M2 Affinity Gel, followed by western blotting with anti-CD109-C-9 mAb (middle panel). Black arrowheads: 180-kDa bands; western blot of IgG light chain is indicated as an internal control. B, Quantitative assessment of concentration of serum tumor-secreted FLAG-hCD109 in HEK293-FLAG-hCD109 xenografted mice after tumor resection using CD109 ELISA (N = 4). BO: Before operation.