4.1 YKL-40 protein expression by stable cell line establishment
The cloned YKL-40/pcDNA 3.1 (Fig 1) was sequenced and multiplied by E.coli. The plasmid was then extract with the plasmid extraction kit, and then transfected into BALB/3T3 cells. Because we use the eGFP plasmid was as positive control and representative of transfection rate, it can be detected by cytofluorescence assay (Fig 3). Different groups of 24 hours, 48 hours, and 72 hours was then selected by treatment with G418 at the concentration of 800 ng/μL. There were totally 15 cell lines survived the selection, but not everyone expression the recombinant YKL-40 protein correctly and successfully. And we discovered that there were two types of the transfected cells (Fig 4), one will scatter evenly and the other will overlapped.
After confirmation and quantification, CL2436, CL4805 and CL7208 were the cell lines that can correctly express the YKL-40 protein. However, CL7208 expresses the YKL-40 protein more rapidly and massively. Therefore it is chosen to be our primary protein expression cell line.
4.2 YKL-40 mRNA expression examination
In order to confirm that the cell lines we used in this study don’t express YKL-40 mRNA or protein spontaneously. We examined all the cell lines for the expression of
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YKL-40 mRNA. The mRNA was examined by extract the total RNA and transcribed into cDNA, and performed polymerase chain reaction (PCR) to check the specific sequence product of YKL-40 mRNA. We tested BALB/3T3, CL2436, CL7208, CMT-1, MPG, and the other canine mammary gland tumor cell line CF41, the result showed no mRNA expressed in these cell lines (Fig 2A). For further investigation, we examined the osteosarcoma cell line D17, lymphoma cell lines CLBL-1, UL-1, CLC, and melanoma cell lines CM-01-01, M1, M2, M3, M4, and M5. The result showed that UL-1, CLC, and M1 don’t express YKL-40 mRNA, while CM-01-01, M2 to M5 expressed YKL-40 mRNA spontaneously and CLBL-1 expressed sparsely (Fig 2B).
4.3 Purification of the recombinant canine YKL-40
The recombinant canine YKL-40 protein was secreted into the cultured medium by CL7208 cells. The cultured medium were collected and concentrated by centrifugal concentrator tubes, the concentration was about 20 folds. We first used the His-column to purify the concentrated recombinant canine YKL-40 protein, the different fluid portion of purification was collected and separated by 10% SDS-PAGE and western blotted, and membrane was probed with anti-His-tag antibody (Fig. 5A).
The result showed that the His-column didn’t bind the recombinant canine YKL-40
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protein successfully, most of the protein was presented in the flow-through fluid and the rest of the protein was washed away after washing buffer applied. No protein was detected in the elute fluid, even after concentration with centrifugal concentrator.
Because YKL-40 has the ability to bind heparin, we used the heparin-column to purify this protein instead (Fig. 5B). No protein was detected in the flow-through fluid and the washed fluid. Most of the protein was successfully bound by the heparin-column and eluted. The protein can still be detected after concentration by using the centrifugal concentrator.
4.4 Proliferation assay
To test if the recombinant canine YKL-40 has the ability to stimulate cell to proliferate, we evaluate the effect of the recombinant canine YKL-40 in CMT-1 (Fig 6A) and MPG cells (Fig 6B). After several times of repeat, the recombinant canine YKL-40 didn’t show any significant effect in the stimulation of cell proliferation.
There seems to be a trend in the effect of the recombinant canine YKL-40, and the cell treated with the recombinant canine YKL-40 do proliferate better than the ones which were not treated. And the result seems to have a dose-dependent pattern in the treatment.
4.5 Migration assay
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To test if the recombinant canine YKL-40 has the ability in stimulation of cell migration, we applied the CMT-1 and MPG cells to the test. After different time of treatment, the difference became apparent after 24 hours. In the group treated with the recombinant canine YKL-40, the cells migrate and recover the gap faster than the control group in a dose-dependent pattern. This effect was more obvious in CMT-1 cells (Fig. 7A) than in MPG cells (Fig 8A), as the result being quantified by computer software (Fig. 7B, 8B). *P<0.05 and **P<0.01 compared with corresponding control cells treated with serum-free DMEM.
4.6 Invasion assay
To examine whether the YKL-40 protein has the ability to stimulate cancer cell invade into the extracellular matrix (ECM), we applied the matrigel to mimic the property of ECM. By observation of the opposite side of the membrane of the insert, we found that the YKL-40 stimulates the invasiveness of the cancer cells in a dose-dependent manner (Fig 9).
4.7 Cell signal pathway analysis
In the result of cell signal pathway investigation, YKL-40 was found to induce the PI3K/AKT pathway both in CMT-1 and MPG cells (Fig 11, 12). The activated form of AKT was increased by YKL-40 in as low as 10 ng/mL, the result in CMT-1 and
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MPG cells both showed a dose-dependent manner. In CMT-1 cells, the activated form of MAPK (ERK1/2) were also induced by YKL-40 in as low as 10 ng/mL and in a dose-dependent manner. However, this result wasn’t showed in MPG cells.
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