SCUBE2 was obtained from OriGene Technologies (Rockville, MD). The SCUBE2 sequence is the same as NM_020974, except nucleotides 1287 to 1526 are spliced out in the clone. Because this commercially available cDNA for human SCUBE2 represents a splice variant lacking a portion of the
EGF-like repeats and spacer region (Yang et al., 2002), Yang et al. swapped in a cDNA fragment to correct the defective region by a standard molecular
biology method. The resulting cDNA encodes a polypeptide (SCUBE2-FL, including the amino acids, 1-1028) composed of an organized protein domain structure consistent with that of its zebrafish orthologue and all other SCUBE protein members (see Figure 1) (Wu et al., 2004; Kawakami et al., 2005;
Hollway et al., 2006; Woods et al., 2005). Other three deletion expression constructs of the SCUBE2 are used in this experiment. Domain organization of these expression constructs is shown in Figure 3 (D4, amino acids 664–1028;
ty97, amino acids 1–659; rw87, amino acids 1–223; SP, signal peptide; E, EGF-like repeats; Cys-rich, cysteine-rich motifs; CUB, CUB domain). Two of these deletion constructs, SCUBE2-ty97 and SCUBE2-rw87 mutant, were made by mimicking two null mutant alleles (ty97 or rw87) in the zebrafish Scube2 (Kawakami et al., 2005; Hollway et al., 2006; Liu et al., 2002). The sequence of SCUBE1 and SCUBE3 are based on the gene prediction and public sequence information (GenBankTM with accession number AF525689 (SCUBE1) and GenBankTM accession number AF452494 (SCUBE3),
respectively). The expression plasmid for prepro BMP2 containing an open reading frame of human BMP2 cDNA (encoding amino acids 1 ~ 398). These expression plasmids were made in the pcDNA3.1 (+) vectors. The
BMP-responsive promoter luciferase reporter construct I-BRE-Luc is described
previously (Benchabane et al., 2003).
The pFLAG-CMV-1 (Sigma) was used to include a FLAG tag at the amino terminus of target protein immediately after the signal peptide sequence at the NH2 terminus for easy detection. The pcDNA4/Myc-His (Invitrogen) was used to add a Myc tag to the carboxyl terminus of target protein.
Cell Culture and Transfection. HEK (human embryonic kidney)-293T, MCF-7-tet off and HepG2 were maintained in DMEM (Dulbecco's minimal essential medium) supplemented with 10 % heat-inactivated FBS (fetal bovine serum), 100 units/ml penicillin and 100 μg/ml streptomycin (GIBCO 15140-122) at 37 °C in an atmosphere of 5 % CO2. Cells were seeded in 6-well plates overnight before transfection HEK-293T were transfected by use of
Lipofectamine™ 2000 (Invitrogen, 51124). The total amount of DNA was kept constant in transfections with empty vectors, SCUBE2-FL, SCUBE2-D4, SCUBE1 and SCUBE3.
Immunoprecipitation and Western Blot Analyses. Two days after transfection, transfected cells were washed once with PBS and lysed for 15 minutes on ice in 0.5 ml of lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100, 25 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin). Lysates were
clarified by centrifugation at 4°C for 15 minutes at 10,000 × g. Cells lysates were incubated with 1 μg of indicated antibody and 20 μl of 50 % (v/v) protein A-agarose (Pierce, Thermo Scientific, Rockford, IL) for 2 hours with gentle rocking. After three washes with lysis buffer, precipitated complexes were solubilized by boiling in Laemmli sample buffer, fractionated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. The membranes were blocked with PBS, pH 7.5, containing 0.1 % gelatin and 0.05 % Tween 20 and were blotted with the indicated antibodies. After two washes, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (The Jackson Laboratories, Maine) for 1 hour. After washing the membranes, the reactive bands were visualized with the enhanced chemiluminescence system (Amersham Biosciences).
For detections of the secretion properties of various SCUBE2 deletion constructs, we collect conditioned medium after 48 hours post-transfection.
And then cells were detached with PBS/EDTA. Samples from conditioned culture medium (Medium) and cell lysates (Cell) were separated by 4-20 % SDS-PAGE and transferred to polyvinylidene difluoride membranes.
Recombinant SCUBE2 proteins and various deletion constructs were detected by Western blotting with anti-Flag M2 antibody.
FACS
TMAnalysis. To verify the cell-surface expression of FLAG-tagged
SCUBE2, transfected cells were collected and suspended in PBS/EDTA, 2 % bovine serum albumin in a volume of 0.25 ml. Either 1 μg of purified anti-FLAG M2 antibody (Sigma, F1804)or or isotype control antibody (IgG1) are added for 45 minutes on ice, Subsequently fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody (1:100 dilution, Jackson ImmunoResearch Laboratories, West Grove, PA) was added and incubated for 45 minutes on ice.
FACSTM analyses were performed with a FACSTM Scan (Becton Dickinson, Mountain View, CA). Histograms weregenerated from measurements of 10,000 cells, and data wereanalyzed by use of the CellQuest software on a FACSTM caliber system.
Establishment of the MCF-7 Breast Tumor Cell Line Stably
Expressing SCUBE2. The MCF-7 Tet-off Vector or MCF-7 Tet-off SCUBE2
cell lines were derived from the stable transfection of MCF-7 Tet-off cells (Clontech Laboratories, Inc.) with an empty pTRE2hyg plasmid (Clontech Laboratories, Inc.), a plasmid encoding the FLAG-tagged full-length (FL) or D4 mutant of human SCUBE2 (FLAG.SCUBE2-FL or -D4), respectively. Stable cell clones were grown in the presence of 10 μg/ml doxycycline (Dox; to suppress SCUBE2 expression) (Sigma, D9891) and selected by resistance to
G418 (100 μg/ml) (GIBCO, 11811-031) and hygromycin (100μg/ml) (Sigma H-3274). Established cell lines were further verified by anti-FLAG western blot analysis to assess Dox-responsive FLAG.SCUBE2 protein expression.
Luciferase Activity Assays. Human HepG2 cells (3 x 10
5 cells per well) were seeded into 24-well plates and transfected on the following day with 0.4 μg of the BMP-inducible luciferase reporter I-BRE-Luc (Benchabane et al., 2003) and 0.01 μg of the Renilla luciferase reporter vector used as an internal control. The transfected responding cells (HepG2) were stimulated withconditional media from signaling cells (HEK-293T) co-transfected with the BMP2 expression plasmid alone or in combination with various SCUBE2 deletion constructs, including the SCUBE2-FL, SCUBE2-D4, SCUBE2-ty97 and SCUBE2-rw87, respectively. Luciferase activity was measured following 24 hours incubation by the use of the dual reporter system (Promega, Madison, WI). Data are expressed as relative luciferase activity (firefly luciferase activity divided by Renilla luciferase activity).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
Cell Proliferation Assay. The effect of SCUBE2 on the proliferation of MCF-7
breast cancer cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Briefly, actively growing MCF-7
Tet-off Vector, MCF-7 Tet-off SCUBE2-FL or -D4 stable cells were trypsinized and plated onto 96-well cell culture plates at 2000 cells/well in 200 μl complete media containing doxycycline. Doxycycline was removed from the medium on the next day to induce gene expression for various times. Each data point was performed in quadruplicate, and the results are presented as relative cell growth (%, mean ± SD).