Antibodies and reagents
Antibodies used were mouse anti-E-cad IgG2a (part #610181), mouse anti-β-catenin IgG1 (610153), mouse anti-CD56 (NCAM13; 556324), anti-CD56 (NCAM 12F8; 556325), anti-STAT3 (610189) from BD Biosciences (San Jose, CA, USA); mouse anti-N-cad IgG1 (sc59987), anti-CK1α (sc6477), anti-c-Myc (sc40) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse anti-VM IgG1 (V5255), mouse anti-PSA-NCAM antibody IgM 5A5 from Developmental Studies Hybridoma Bank (University of Iowa, IA, USA); anti-β-tubulin I IgG1 (T7816), anti-FN (F3648) from Sigma-Aldrich (St. Louis, MO, USA); anti-EGFR (D38B1), anti-p-EGFR (Tyr 1068; D7A5), anti-p-STAT3 (Tyr 705; D3A7), anti-GSK-3β (27C10), anti-slug (C19G7), anti-histone H3 (D1H2) from Cell Signaling Technology (Beverly, MA, USA); horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (A0216), HRP-labeled goat anti-rabbit IgG (A0208), FITC-labeled goat anti-mouse IgG (A0568), Cy3-labeled goat anti-mouse IgG (A0521), donkey anti-goat IgG (A0181), anti-His-tag mAb (AH367) from Beyotime Institute of Biotechnology (Haimen, China); FITC-labeled goat anti-rabbit IgG (CW0114), goat anti-rat IgG (CW0166) from CWBIO (Beijing, China).
FN, laminin, collagen IV, puromycin, and hygromycin were from Sigma-Aldrich. Matrigel was from Corning Life Sciences (Tewksbury, MA, USA). Other reagents were from Sigma unless described otherwise.
Cell lines and cell culture
Mouse mammary epithelial cell line NMuMG and human breast epithelial cell line MCF10A were from American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in DMEM (Hyclone; Logan, UT, USA) supplemented with 10% FBS (Hyclone), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Gibco; Carlsbad, CA, USA) in a humidified 5% CO2 atmosphere at 37 °C. For NMuMG, medium was supplemented with 10 μg/mL insulin (Sigma). For MCF10A, medium was supplemented with 1% sodium pyruvate (Solarbio; Beijing).
Patients and tissue samples
BC tissues and normal breast tissues were obtained from the First Affiliated Hospital of Xi’an Jiaotong University. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki. Experiments using human tissues were approved by the Research Ethics Committee of Northwest University.
Gene amplification and transfection
Mouse genes NCAM-120, NCAM-140, and STX were amplified by PCR as previously described[20]. Briefly, primers were as follows: for NCAM-120: sense 5'-CCCAAGCTTGCCACCATGCTGCGAACTAAGGATC, antisense 5'-CCGCTCGAGTCAGAGCAGAAGAAGAGTCAC; for NCAM-140: sense 5'-CCCAAGCTTGCCACCATGCTGCGAACTAAGGATC, antisense 5'-CCGCTCGAGCGGTCATGCTTTGCTCTCATTC; for STX: sense 5'-GAAGGCCTGCCACCATGCAGCTGCAGTTCCG; antisense (containing the codons for 6His residues) 5'-CTAGCTAGCTTAGTGGTGGTGGTGGTGGTGCGTAGCCCCATCACACT. PCR products were ligated into vector pcDNA3.1 (Invitrogen; Carlsbad, CA, USA) or pIREShyg3 (Clontech; Mountain View, CA, USA), respectively. Stably transfected NMuMG cells were selected with G418 or hygromycin.
Semi-quantitative PCR and quantitative real-time PCR
Semi-quantitative PCR and quantitative real-time PCR was performed as previously described[20]. Briefly, total RNA was isolated using a RNApure Tissue Kit (CWBIO) and reverse-transcripted to cDNA using a ReverTra Ace-α Kit (Toyobo; Osaka, Japan). Primers were designed as shown in Suppl. Table 1. Quantitative real-time PCR (RT-qPCR) was performed with UltraSYBR Mixture (CWBIO) using a CFX96 PCR detection system (Bio-Rad; Richmond, CA, USA). Gene expression was quantified by the 2-ΔΔCt method[21] and expressed as mean ± SD from triplicate experiments.
Migration assay
Migration assay was performed as previously described[20]. Cells (5 × 104) were plated in an upper transwell insert (8-μm pore size, Corning) in serum-free medium, and complete-medium was added to the bottom chamber. After 24 h culture, cells migrating across the membrane were stained with 0.1% crystal violet, and photographed under microscope (magnification of 100×).
Wound closure assay
NMuMG cells were plated at a high density in 6-well plates and incubated until a confluent monolayer was achieved. A scratch was made with 100 μL pipette tips in each well and cultures were washed with PBS to remove any cell debris. Cells were incubated in DMEM supplementd with 10% FBS and 5 μg/mL mitomycin C (Sigma-Aldrich) for 24 h. Cell migration between the scratch areas was monitored at 0 h and 24 h, using optical microscope. Migration distance was measured with Image Pro Plus 6.0 (Media Cybemetics, MD, USA)
Proliferation (MTT) assay
Cell proliferation was determined by MTT assay as described previously[22]. Briefly, cells (4 × 103/well) in 96-well plates were incubated 4 h with 4 μL MTT solution (Cers, Yantai city, China). The reaction was terminated by addition of 100 μL DMSO, and absorbance at 595 nm was determined.
Cell motility assay
Cell motility was determined by phagokinetic gold sol assay as described previously[23]. Cells (2 × 103) in complete culture medium were seeded onto gold sol-coated wells, incubated for 12-18 h, and photographed under an inverted microscope. Tracking areas of 50 cells were measured using the ToupView imaging system and expressed as square pixels.
Cell adhesion assay
Adhesion assays were performed as described previously[24]. In brief, 96-well plates were coated overnight at 37 °C with FN (1 μg/well), collagen IV (1.5 μg/well), Matrigel (80 μg/well), or laminin (1 μg/well). Wells were rinsed and blocked for 1 h with 1% BSA in Hank’s balanced salt solution (HBSS) at 37 °C. Cells were harvested with trypsin, plated (40,000 cells per coated well), and incubated 30 min at 37 °C. Wells were washed gently with HBSS to remove unattached cells. Adherent cells were fixed with 4% paraformaldehyde for 10 min, stained with 0.1% crystal violet (in 20% methanol) for 10 min, dissolved in 100 μL of 10% acetic acid after removal of excess dye with PBS, and absorbance was measured at 595 nm.
Immunofluorescence staining
Immunofluorescence staining was performed as previously described[20]. Cells (2 × 104) coated on glass cover slips in 24-well plates, were washed with PBS, fixed with 4% fresh paraformaldehyde, blocked with 1% BSA, incubated with appropriate antibody and Hoechst 33342 (Invitrogen; Paisley, UK), mounted with Glycergel (Dako; Carpinteria, CA, USA), and observed by fluorescence microscopy (model Eclipse Ti-U, Nikon; Tokyo, Japan) at 600× magnification.
Western blotting
Western blotting was performed as previously described[20]. Equal amounts of proteins were loaded on SDS-PAGE gels and transferred onto a PVDF membrane. The membrane was blocked with 5% BSA, incubated with primary antibody and appropriate HRP-conjugated secondary antibody, visualized by Pro-Light HRP (Tiangen Biotech; Beijing), and photographed using a Molecular Imager ChemiDoc XRS+ system (Bio-Rad).
Flow cytometry
Cells were plated in triplicate on 24-well plates (2×105 cells/well) as described previously[25], detached, incubated with primary and secondary FITC-conjugated antibody. Signals from cells were detected by flow cytometry (FACSCalibur, BD; San Jose, CA, USA), with data acquisition and analysis by the FlowJo software program (Tree Star; San Carlos, CA, USA).
Gene silencing with small interfering RNA (siRNA)
Duplexes of 21 nucleotides of mouse STX siRNA target sequence and negative control siRNA (NC), having no homology to other known mouse genes, were designed and synthesized by Invitrogen; for mouse STXi, 5’-GCCUGGAGAUAUUAUUCAUTT (sense). siRNA was transfected using Lipofectamine 2000 reagent, and cells were examined after 24 h. Suppression of STX expression was verified by semi-quantitative and quantitative RT-PCR.
Luciferase reporter assay
β-Catenin transcription was assessed using TOP FLASH/ FOP FLASH reporter luciferase assay[26]. Cells were seeded onto 24-well plates and transfected using Lipofectamine 2000 (Invitrogen) with 1 μg M50 Super 8X TOP Flash (Plasmid 12456) or M51 Super 8X FOP Flash (Plasmid 12457) reporter vector (Addgene), together with 0.05 μg internal pRL-TK Renilla plasmid (Promega). Cells were processed 48 h after co-transfection for luciferase reporter activity using a Dual Luciferase Reporter System (Promega). Firefly luciferase activity was normalized against Renilla luciferase activity. Reporter assay results were presented as relative luciferase activity (averaged ratio of firefly/Renilla luciferase ± S.E.) from three or more independent experiments.
Immunoprecipitation analysis
Cells were cultured on 60-mm diameter plates, washed with PBS, and added with IP lysis buffer (Beyotime), incubated on ice for 30 min, and centrifuged at 14,000 g for 15 min at 4 °C. Supernatants were collected. Proteins (500 μg) was incubated with 2 μg primary antibody for 1 h at 4°C, incubated with 20 µL of Protein A/G PLUS-Agarose (sc-2003, Santa Cruz) at 4°C for overnight, centrifuged at 2,000 g for 5 min at 4°C, washed to remove nonspecific binding with PBS. SDS-PAGE loading buffer was added, and the samples were boiled at 100°C for 15 min. Immunoprecipitated proteins were then subjected to western blot as described above.
Immunohistochemistry
Tissue slides were dewaxed, rehydrated, antigen retrieval, and incubated with 3% hydrogen peroxide for 30 min and blocked in 10% normal mouse serum for 30 min. The slides were then incubated with primary antibodies against NCAM (1:1000; Santa Cruze) at 4°C for overnight. The slides were rinsed with PBS, incubated with HRP-conjugated secondary antibody, visualized with DAB (Sigma-Aldrich).
Data analysis
Data were statistically analyzed using the GraphPad Prism (GraphPad software; San Diego, CA, USA). Differences between means were evaluated by Student's t-test, and p-values <0.05 were considered significant.