3.1 Experimental animals
All experimental beagle dogs had be de-wormed and vaccinated with distemper, adenovirus, parainfluenza virus, leptospirosis, parvovirus, coronavirus, infectious hepatitis virus, and hemorrhagic icterus virus in the eight in one shot vaccine. All experiments were performed according to the National Taiwan University Animal Experimental Ethics Committee guidelines.
3.2 Cell lines
In this study, we used two different canine mammary gland tumor cell lines, CMT-1 and MPG, which were gifts from Dr. Lin’s laboratory (Chang et al. 2010), to perform biological functional assays. Both CMT-1 and MPG were cultured in the complete Dulbecco’s Modified Eagle Medium (DMEM, Caisson, USA) supplemented with 10% fetal bovine serum (FBS, Caisson, USA) and 1% antibiotics (Casisson, USA).
These two cell lines don’t spontaneously express YKL-40 mRNA and protein. In expression of the YKL-40 protein, we applied the BALB/3T3 cells in the transfection. The BALB/3T3 (ATCC®CCL-163.2TM) cell line was from the American Type Culture Collection (ATCC). After transfection and selection, we established the stable cell line CL7208 which can stably, continuously, and
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spontaneously express the recombinant canine YKL-40 protein.
3.3 Anitbodies
In this study, we used the mouse source anti-6x-Histidine monoclonal antibody (mAb) (Novus Biologicals, CO, USA) to probe the recombinant canine YKL-40 protein in Western blot analysis, dilution 1:2000. In the cell signal pathway analysis, we used the following antibodies to probe the cell signal pathway proteins in the Western blot: rabbit source anti-phospho-Akt mAb (#4060, Cell Signaling, MA, USA), dilution 1:2000; rabbit source anti-Akt antibody (#9272, Cell Signaling, MA, USA), dilution 1:1000; rabbit source anti-phospho-MAPK (Erk1/2) mAb (#4370, Cell Signaling, MA, USA), dilution 1:2000; mouse source anti-MAPK (ERK1/2) mAb (#4696, Cell Signaling, MA, USA), dilution 1:2000; rabbit source anti-phospho-Stat3 mAb (#9145, Cell Signaling, MA, USA), dilution 1:2000;
mouse source anti-Stat3 mAb (#9139, Cell Signaling, MA, USA), dilution 1:1000.
3.4 Total RNA extraction
In the YKL-40 cloning portion, we used TRIzolTM (Invitrogen) to extract total RNA from canine macrophages, and followed the procedure suggested by the manufacturer. While in the YKL-40 mRNA expression examination for the canine mammary gland tumor cells, we used RNAzol® RT (Molecular Research Center,
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Inc., OH, USA) to extract total RNA from the cMGT cells.
3.5 RT-PCR
The RNA was subsequently reverse-transcribed into cDNA with a Super-Script II reverse transcriptase kit (Invitrogen) according to the manufacturer’s suggestion.
Because that so far only partial canine YKL-40 sequence was predicted (Accession number: XM_547343, GenBank database: http://www.ncbi.nlm.nih.gov/genbank/), we obtained the full length of the canine YKL-40 gene for the plasmid insertion. The total RNA will first be quantified and qualified by using a spectrometer, then 1 μg of RNA was proceeded with a de-genomic-DNA procedure: using RQ1 RNase-free DNase (Proméga, Madison, WI, USA), and RQ1 DNase 10X Reaction Buffer (Proméga, Madison, WI, USA). After incubation at 37℃ for 30 minutes, the RQ1 Stop Solution (Proméga, Madison, WI, USA) was added to stop the DNase reaction at 65 ℃ for 10 minutes. The RNA without genomic-DNA was then reverse-transcribed into cDNA by using the HiScript I Reverse Transcriptase (MmLV) (Bionovas, Toronto, Ontario, Canada), 5X Strand Buffer (Bionovas, Toronto, Ontario, Canada), 100 mM DTT (Bionovas, Toronto, Ontario, Canada), Fermentas RiboLock RNase Inhibitor (Thermo Scientific, Burlington, Ontario, Canada), 1μM oligo dT primer and random primer. The PCR protocol as following:
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30℃,10 min; 42℃, 60 min; 70℃ 15 min.
3.6 Preparation and propagation of YKL-40/pcDNA 3.1 plasmid
The canine YKL-40 gene without stop codon (InC) was obtained from RT-PCR using primer pair F and InR, and the product InC was subsequently cloned into pcDNA3.1/V5-His-TOPO plasmid using the pcDNATM3.1/V5-His-TOPO®TA Expression Kit (Invitrogen, CA, USA), and followed the procedure suggested by the manufacturer. Once the canine YKL-40 gene is cloned into the pcDNA 3.1/V5-His-TOPO plasmid, it is transformed into E.coli for propagation. The multiplied plasmid is then extract with the Plasmid Midi Kit (Qiagen), and follow the procedure suggested by the manufacturer.
3.7 Transfection of canine YKL-40 gene into BALB/3T3 cells
This extract canine YKL-40/pcDNA 3.1 plasmid was then transfected into BALB/3T3 cells by using Lipofectamine 2000 (Invitrogen, CA, USA); the plasmid pcDNA 3.1 was used as a mock control, while the eGFP plasmid was used as a positive control and representative of the transfection rate. The transfected cells were harvested after 24, 36 and 72 hours. 1000 cells from each time point were transferred to 96-well plate and G418 (Amresco, OH, USA) at the concentration of 800 ng/μL was added into each well to select the successfully transfected cell. Since
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the pcDNA3.1 plasmid contains an anti-neomycin gene, successfully transfected cell will survive in the presence of G418. By using this method, we obtained several cell lines which were developed from single cell. After 2 weeks of treatment of G418, the concentration was decreased to 400 ng/μL, for the maintenance growth of the cell line.
3.8 Expression of canine YKL-40 protein in BALB/3T3 cells
The transfected cell lines were named by the harvesting time, There were totally 15 cell lines acquired, after confirmation and quantification, we chose the cell line CL7208 to produce our recombinant YKL-40 protein. The protein was overexpressed by this cell line, and secreted into the medium. The 0% FBS medium was used to collect the secreted protein.
3.9 Concentration of the recombinant canine YKL-40 protein
The recombinant canine YKL-40 is a secreted glycoprotein, which will be secreted into the culture medium. We collect the FBS-free DMEM cultured with CL7208 cells after 24, 48 and 72 hours of incubation. Once the cultured medium is collected, the proteinase inhibitor (Bionovas, Toronto, Ontario, Canada) is added follow the manufacturer’s suggestion. The medium will be concentrated with the centrifugal concentrator tubes (Vivaspin 20, pore size: 10 kDa MWCO, GE Healthcare, Little
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Chalfont Buckinghamshire, UK), and centrifuged with 2330 xg at 4℃ (Kubota 5910 centrifuge, Japan) first. The centrifuge time depends on the volume of the medium needs to be concentrated, 30 minutes for one centrifugation, mostly 3 to 5 hours at total. The protein was concentrated about 20-40 folds, and the medium will be replaced by phosphate buffered saline (PBS). The concentrated protein will be quantified after concentration is completed. After protein quantification, we found that the most appropriate time for collection is 48 to 72 hours, because that the protein will reach a certain concentration and the condition of cells still remains well.
3.10 Purification of the recombinant canine YKL-40 protein
The solution was then purified by His-column (IMAC FF 5 mL, GE Healthcare Bio-Sciences, NJ, USA), and Heparin-column (HiTrap Heparin HP 1 mL, GE Healthcare Bio-Sciences, NJ, USA). The different proportion of the purification, including supernatant, flow-through, wash, and elute were collected for protein detection.
3.11 Quantification of the recombinant canine YKL-40 protein
After purification, the eluted and concentrated recombinant canine YkL-40 protein will be quantified with the Coomassie Plus – The Better Bradford AssayTM Kit
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(Thermo Scientific, IL, USA), and followed the procedure suggested by the manufacturer. The bovine serum albumin (BSA) standard (2 mg/mL, Thermo Scientific Pierce, IL, USA) was used to generate a standard curve. Each concentration of protein is triplicate.
3.12 Identification of the purified recombinant canine YKL-40 protein
The purified and concentrated recombinant canine YKL-40 protein will be separated by 10% SDS-PAGE and western blotted. The membrane was probed with mouse source anti-His-tag monoclonal antibody.
3.13 Western blot analysis
The protein samples were boiled with 5X protein loading dye for 10 minutes. The samples are separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with 80 volt for 120 to 150 minutes, and then Western blotted to the polyvinylidene difluoride (PVDF) membranes, the condition is 300 mA for 1 hour. 20 mL of skim milk is used to block the membrane on a rotator shaker at 4℃ for 2 hours. Then the primary antibody will be diluted with the skim milk at a proper dilution ratio and began to probe with the incubation on a rotator shaker at 4℃ overnight. Before adding the secondary antibody, the membrane will be washed 3 times with Tris buffered saline and Tween 20 (TBST), the Tween concentration is
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1% and each washing time is 10 minutes. Then the secondary antibody will be diluted with the skim milk at a proper dilution ratio and began incubation on a rotator shaker at room temperature for 6 hours. The membrane will be washed with TBST three times, each time for 10 minutes. After washing, the membrane will be put on a plastic board and add the enhanced chemiluminescence (ECL, RapidStep™
ECL Reagent) (Millipore, MA, USA). The membrane will be photographed by the image system Geliance 600 (Perkin Elmer, MA, USA).
3.14 Proliferation assay
The cMGT cell lines CMT-1 and MPG were seeded into 96-well plates at 3000 to 5000 cells per well density, and allowed to adhere for 24 hours in DMEM containing 10% FBS. The medium was then discarded and washed with FBS-free DMEM. The cells were then divided into six groups, treated with DMEM plus 10, 100, and 1000 ng/mL of YKL-40. The medium DMEM without FBS cultured with BALB/3T3 and CL7208 cells were also used to this test. DMEM without FBS was used as the negative control. Each condition was assayed in triplicate wells.
3.15 Migration assay
The cMGT cell lines CMT-1 and MPG were seeded into 6-well plates at 2x105 cells per well density. After 24 hours of incubation with 10% FBS DMEM, each well was
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grooved with 1 mL pipette tip. The cultured medium was discarded and the wells were then washed by DMEM without FBS. The purified YKL-40 protein in different concentration was added to each well. The result was observed at 18, 24, 36, and 48 hours with microscope. The results were then calculated by computer software and also analyzed.
3.16 Invasion assay
Invasion assays were conducted in cell culture inserts (BD Falcon, NJ, USA) with a transparent PET track-etched membrane (pore size 8.0 μm). 100 μL of Matrigel (BD, NJ, USA) was added into the insert at 4℃ and incubate at 37℃ for gelling, then 4x105 cells in 200 μL of DMEM without FBS were added onto the gel. This prepared insert was then placed into the 24-well plate, each well contains different medium to be tested.
3.17 Autoantigen analysis
The recombinant canine YKL-40 protein was separated by 10% SDS-PAGE and western blotted. The membrane was then probed with the dog serum from the healthy dog without any known disease of our experimental Beagles. The sera were diluted 500 times for the use as primary antibody in the Western blot analysis. Then the anti-canine antibody conjugated with horseradish peroxidase (HRP) was used a
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secondary antibody to probe the bound serum antibodies. The membrane was observed with ECL and photographed by the image system as well as the Western blot analysis.
3.18 Cell signal pathway analysis
This analysis applied the canine mammary gland tumor cell lines CMT-1 and MPG to the test. After trypsinized from the 175 cm2 flask, the cells are distributed into 5 eppendorfs per 5x106 for each tube. Then centrifuge with 1500 xg for 10 minutes (Prism Microcentrifuge) (Labnet International, Inc., NJ, USA), and discard the medium. 1 mL of DMEM without FBS will added into each eppendorf and incubate at 37℃ for 1 hour serum starvation. The cells will be treated with 10% FBS DMEM, 10, 100, 1000 ng/mL of recombinant YKL-40 protein, respectively, the DMEM without FBS will be the negative control. After 1 hour of treatment, the cells will be centrifuged with 1500 xg for 10 minutes, and discard the medium. Then 100 μm of RIPA lysis buffer (Millipore, MA, USA), which contains 1% of proteinase inhibitor (Bionovas, Toronto, Ontario, Canada), 2 % of phosphatase inhibitor (Sigma-Aldrich, MO, USA), and 1% of Na3VO4, will be added into each eppendorf. After 30 minutes of incubation on ice, with vortex thoroughly every 5 minutes, and centrifuge with 3000 xg for 10 minutes. The supernatant will be transfer to a new eppendorf and the
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total protein will be quantified. 100 μg of protein will be applied to the Western blot, the membrane will be probed with anti-phospho-AKT, anti-phospho-MAPK (p-ERK1/2) and anti-phospho-STAT3 antibodies. Then the membrane will be stripped and probed with anti-AkT, anti-MAPK, anti-STAT3 and anti-actin antibodies again.
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