Cell culture & bacteria
F11 cells was purchased from European Collection of Authenticated Cell Cultures (ECACC, 08062601) and was cultured in DMEM supplemented with 10% FBS (both Invitrogen) in a humidified atmosphere at 37 ℃ with 5% CO2. Escherichia coli DH5α strain was cultured at 37°C in Luria-Bertani (LB) broth medium supplied with kanamycin (50 ug/ml) and was used as a host in transformation. The institutional review board of Kaohsiung Medical University, Kaohsiung, Taiwan approved gene recombinant experiment in this study (KMU-106076).
pAAV-GlyRα1, pAAV-GlyRα3 recombinant vector construction
Firstly, total RNA was extracted from brain of SD rat, then was reversed to cDNA by using MMLV Reverse Transcription kit (Protech). GlyRα1, GlyRα3 fragments were PCR-amplified from rat cDNA using primers as following. Primers for GlyRα1 (forward: 5’-ACAGCGGCCGCACCATGTACAGCTTCAACACTCTG-3’, reverse: 5’-GGCGATATCTCACTTGTTGTGGACGTC-3’); primers for GlyRα3 (forward: 5’-ACAGCGGCCGCACCATGCCTTGGATAAGACTG-3’, reverse: 5’-GGCGATATCTTAATCTTGCTGATGATGAATG-3’). These two GlyRα fragments of PCR products were eluted and purified from Low Melting Point Agarose gels (ThermoFisher Scientific), then were ligated into pCR™-Blunt II-TOPO® vector by Zero Blunt® TOPO® PCR Cloning Kits (Invitrogen) to construct recombinant vectors pBlunt-GlyRα1, pBlunt-GlyRα3. Later, these two recombinant vectors were transfer to Escherichia coli DH5α competent cells by transformation. Extraction and purification of recombinant vectors pBlunt-GlyRα1, pBlunt-GlyRα3 from Escherichia coli DH5α cells was carried out with Presto™ Mini Plasmid Kit (PDH100, PDH300) (Geneaid, Taiwan). Accuracy of GlyRα1, GlyRα3 fragments which ligated with pCR™Blunt II-TOPO® vector was further confirmed by PCR (primers for GlyRα1 forward: 5’-AAGAATTTCCCGATGGACGTA-3’, reverse: 5’-GTAGTGCTTGGTGCAGTA-3’; primers for GlyRα3 (forward: 5’-AAACACTACAATACAGGAAAGTTTAC-3’, reverse: 5’-CAGTGGTGATACCCAACG-3’) and DNA sequencing. Secondary, Not I and EcoRV restriction enzymes were selected to cleaves pBlunt-GlyRα1, pBlunt-GlyRα3 recombinant DNA and pAAV-IRES-GFP expression vector. The restriction products were detected by Low Melting Point Agarose gels electrophoresed, eluted, purified and subjected to ligation reaction by T4 DNA ligase (BioLabs) to construct recombinant vectors pAAV-GlyRα1, pAAV-GlyRα3 (Fig. 1). Lastly, by transformation, these two recombinant vectors were then transfer to Escherichia coli DH5α competent cells. Recombinant vectors pAAV-GlyRα1, pAAV-GlyRα3 from Escherichia coli DH5α cells was extracted and purified again with Presto™ Mini Plasmid Kit (PDH100, PDH300) (Geneaid, Taiwan). Accuracy of GlyRα1, GlyRα3 fragments from pAAV-GlyRα1, pAAV-GlyRα3 were further confirmed by PCR (primers for GlyRα1 forward: 5’-AAGAATTTCCCGATGGACGTA-3’, reverse: 5’-GTAGTGCTTGGTGCAGTA-3’; primers for GlyRα3 (forward: 5’-AAACACTACAATACAGGAAAGTTTAC-3’, reverse: 5’-CAGTGGTGATACCCAACG-3’) and DNA sequencing. The correct recombinant clones which containing pAAV-GlyRα1, or pAAV-GlyRα3 were store at -80℃ for further use.
pAAV-GlyRα1, pAAV-GlyRα3 transfection into F11 cells
Seeded F11 cells into a 6 well plate at a density of 3x105 cells/well. When cell confluence reached 70%, transfected with pAAV, pAAV-GlyRα1 or pAAV-GlyRα3 (2 ug or 5 ug) recombinant vectors with a Lipofectamine® 2000 Transfection Reagent (Invitrogen) at 37˚C with 5% CO2 for 24, 48, 72 hours, respectively. Transfection efficiency was assayed by counting green fluorescent protein (GFP) emitted from pAAV-GlyRα1, or pAAV-GlyRα3 recombinant vectors inside the F11 cells.
Cell Viability assay
The effect of pAAV, pAAV-GlyRα1 or pAAV-GlyRα3 as well as PGE2 on F11 cells was determined by MTT assay. F11 cells (7 x104 cells/well) were seeded in the 24 well plate, after 24 hours culture, 2 ug pAAV, pAAV-GlyRα1, pAAV-GlyRα3 or lipofectamine only was used to transfect F11 cells for 48 hours. In addition, F11 cells treated with Prostaglandin E2 (PGE2, 100 uM) for 60 mins was included in the experiment. In the end, cell viability was assessed by the MTT assay kit (abcam) according to the manufacturer’s protocol. Briefly, remove culture medium and MTT reagent was added into each well, incubated for 2~6 hours at 37ºC, remove MTT reagent then DMSO was added and incubated for 5 minutes. Supernatant was collected and absorbance was measured at OD 550~600 nm. F11 cells without transfection and lipofectamine treatment was used as control.
To survey the effect of PGE2, or recombinant pAAV-GlyRα1 or pAAV-GlyRα3 on inducing pERK phosphorylation and activating transcription factor 3 (ATF-3) activation, 2 ug pAAV, or pAAV-GlyRα1 or pAAV-GlyRα3 was selected to transfect F11 cells (3 x105 cells/well), which seeded into the 6 well plate, for 48 hours. Serum free medium was replaced for another 24 hours, then F11 cells was treated with PGE2 (100 uM) for 5, 15, 30 and 60 mins. F11 cells treated PGE2 alone or F11 cells transfected with pAAV, or pAAV-GlyRα1 or pAAV-GlyRα3 alone was also included in the present study. To investigate the pathway of PGE2 induced pERK phosphorylation, a glycine receptor antagonist Strychnine, EP2 receptor antagonist PF-04418948 was added for 24 hours. One hours after PGE2 administration cell pellets were harvested and pERK phosphorylation was measured. In western blotting, seeded cells as described above were harvested and were homogenized in RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40, and 0.5% sodium deoxycholate) containing protease inhibitor cocktail (Roche, Germany). The protein concentration was determined using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA). 20 ug of total protein was loaded into 8% (w/v) sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The filters were incubated with rabbit monoclonal anti-Phospho-p44/42 MAPK (Erk1/2) (Cell Signaling Technology, 4370p), rabbit monoclonal anti-p44/42 MAPK (Erk1/2) (Cell Signaling Technology, 4695p), rabbit monoclonal anti-phospho-p38 MAPK (mitogen-activated protein kinase; Cell Signaling Technology, Boston, MA, USA), rabbit monoclonal anti-p38 MAPK (Cell Signaling Technology), rabbit anti-ATF3 (NOVUS, NBP1-85816, USA), or mouse monoclonal anti-actin (MAB1501; Indianapolis, IN, USA) primary antibodies. This was followed by reaction with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology). The intensity of each band was visualized with ECL Western blotting detection reagents (Amersham Biosciences, Tokyo, Japan). Protein expression was normalized using β-actin as an internal control and against the protein levels of the control group.
Measurement of cytokines by ELISA
To investigate the possible role of glycine receptors on modulating lipopolysaccharides (LPS) or complete Freund's adjuvant (CFA) induced cell inflammatory reaction. According to the transfection efficiency assay described above, 2 ug pAAV, pAAV-GlyRα1 or pAAV-GlyRα3 was selected to transfect F11 cells (3 x105 cells/well), which seeded into the 6 well plate, for 48 hours. Serum free medium was replaced for another 24 hours, then was treated with LPS (100 ng), or CFA (100 ng) for another 6 hours. Supernatant was collected for analyses of cytokines, including IL-1ß, tumor necrosis factor (TNF)-α, and IL-6 by ELISA (R&D Inc., Minneapolis, MN, USA). F11 cells without pAAV, pAAV-GlyRα1, or pAAV-GlyRα3 transfection or LPS/CFA treatment was used as control.
Results are presented as mean ± SE. Analytical statistics were performed using the SPSS (version 20) software package. Statistical significance was calculated by nonparametric Mann-Whitney U test and for pair-wise comparisons only. In some cases, ANOVA followed by Scheffe multiple post hoc test were used. Differences were considered statistically significant at *p < 0.05, **p<0.01, ***p<0.001.