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Fluorescence intensity of the various fluorescent proteins. Relative fluorescence units (RFU) were measured for cells transfected with AcGFP (A) and (B), DsRed (C) and (D), mStrawberry (E) and (F). Panels (A), (C), and (E) show the RFUs in MH7A cells, and (B), (D) and (F) represent those in C2C12 cells. Open bars and black bars represent 0.1 and 0.8 μg plasmid-transfected cells, respectively. Each result is expressed as the mean ± SD of triplicate experiments. The fluorescence of the control cells harboring pcDNA3.1(+) was subtracted from mean values.
Source publication
Here, we investigate the appropriate fluorescent proteins for use in the culture of synovial MH7A and myoblast C2C12 cells. Fluorescent signal intensities of 3 different fluorescent proteins were examined in these cell lines. The fluorescent intensity of transiently expressed AcGFP, DsRed, and mStrawberry were examined in these cell lines, and the...
Contexts in source publication
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... fluorescence intensities of MH7A cells and C2C12 cells expressing AcGFP (Figure 2A and B) and mStrawberry ( Figure 2E and F), which showed bright fluorescence under microscopy, were much higher than that of the DsRed-monomer ( Figure 2C and D). For in- stance, at 48 h (the time at which images shown in Figure 2 were taken), the fluorescence of AcGFP and mStrawberry in MH7A cells transfected with 0.8 μg of either plasmid (Figure 2A and E) was 5.0-fold and 9.9-fold higher than that of the DsRed-monomer ( Figure 2C). ...
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... fluorescence intensities of MH7A cells and C2C12 cells expressing AcGFP (Figure 2A and B) and mStrawberry ( Figure 2E and F), which showed bright fluorescence under microscopy, were much higher than that of the DsRed-monomer ( Figure 2C and D). For in- stance, at 48 h (the time at which images shown in Figure 2 were taken), the fluorescence of AcGFP and mStrawberry in MH7A cells transfected with 0.8 μg of either plasmid (Figure 2A and E) was 5.0-fold and 9.9-fold higher than that of the DsRed-monomer ( Figure 2C). ...
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... fluorescence intensities of MH7A cells and C2C12 cells expressing AcGFP (Figure 2A and B) and mStrawberry ( Figure 2E and F), which showed bright fluorescence under microscopy, were much higher than that of the DsRed-monomer ( Figure 2C and D). For in- stance, at 48 h (the time at which images shown in Figure 2 were taken), the fluorescence of AcGFP and mStrawberry in MH7A cells transfected with 0.8 μg of either plasmid (Figure 2A and E) was 5.0-fold and 9.9-fold higher than that of the DsRed-monomer ( Figure 2C). ...
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... fluorescence intensities of MH7A cells and C2C12 cells expressing AcGFP (Figure 2A and B) and mStrawberry ( Figure 2E and F), which showed bright fluorescence under microscopy, were much higher than that of the DsRed-monomer ( Figure 2C and D). For in- stance, at 48 h (the time at which images shown in Figure 2 were taken), the fluorescence of AcGFP and mStrawberry in MH7A cells transfected with 0.8 μg of either plasmid (Figure 2A and E) was 5.0-fold and 9.9-fold higher than that of the DsRed-monomer ( Figure 2C). Similarly, in C2C12 cells, fluorescence of AcGFP and mStrawberry ( Figure 2B and F) were 5.0-fold and 8.7-fold higher than that of the DsRed-monomer ( Figure 2F). ...
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... fluorescence intensities of MH7A cells and C2C12 cells expressing AcGFP (Figure 2A and B) and mStrawberry ( Figure 2E and F), which showed bright fluorescence under microscopy, were much higher than that of the DsRed-monomer ( Figure 2C and D). For in- stance, at 48 h (the time at which images shown in Figure 2 were taken), the fluorescence of AcGFP and mStrawberry in MH7A cells transfected with 0.8 μg of either plasmid (Figure 2A and E) was 5.0-fold and 9.9-fold higher than that of the DsRed-monomer ( Figure 2C). Similarly, in C2C12 cells, fluorescence of AcGFP and mStrawberry ( Figure 2B and F) were 5.0-fold and 8.7-fold higher than that of the DsRed-monomer ( Figure 2F). ...
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... fluorescence intensities of MH7A cells and C2C12 cells expressing AcGFP (Figure 2A and B) and mStrawberry ( Figure 2E and F), which showed bright fluorescence under microscopy, were much higher than that of the DsRed-monomer ( Figure 2C and D). For in- stance, at 48 h (the time at which images shown in Figure 2 were taken), the fluorescence of AcGFP and mStrawberry in MH7A cells transfected with 0.8 μg of either plasmid (Figure 2A and E) was 5.0-fold and 9.9-fold higher than that of the DsRed-monomer ( Figure 2C). Similarly, in C2C12 cells, fluorescence of AcGFP and mStrawberry ( Figure 2B and F) were 5.0-fold and 8.7-fold higher than that of the DsRed-monomer ( Figure 2F). ...
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... in- stance, at 48 h (the time at which images shown in Figure 2 were taken), the fluorescence of AcGFP and mStrawberry in MH7A cells transfected with 0.8 μg of either plasmid (Figure 2A and E) was 5.0-fold and 9.9-fold higher than that of the DsRed-monomer ( Figure 2C). Similarly, in C2C12 cells, fluorescence of AcGFP and mStrawberry ( Figure 2B and F) were 5.0-fold and 8.7-fold higher than that of the DsRed-monomer ( Figure 2F). The reason why the DsRed- monomer fluoresces less intensely compared with the other fluorescent proteins is not related to the molecular size of the protein, but rather to its amino acid sequence. ...
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... in- stance, at 48 h (the time at which images shown in Figure 2 were taken), the fluorescence of AcGFP and mStrawberry in MH7A cells transfected with 0.8 μg of either plasmid (Figure 2A and E) was 5.0-fold and 9.9-fold higher than that of the DsRed-monomer ( Figure 2C). Similarly, in C2C12 cells, fluorescence of AcGFP and mStrawberry ( Figure 2B and F) were 5.0-fold and 8.7-fold higher than that of the DsRed-monomer ( Figure 2F). The reason why the DsRed- monomer fluoresces less intensely compared with the other fluorescent proteins is not related to the molecular size of the protein, but rather to its amino acid sequence. ...
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... for the amount of plasmid used in transfection of both cell lines, a greater amount results in higher fluorescence intensity, except for expression of AcGFP in MH7A cells (Figure 2A). In MH7A cells expressing AcGFP, higher fluorescence intensities were observed on transfected cells with 0.1 μg plasmid than cells with 0.8 μg plasmid at 72 h and 96 h. ...
Citations
... Cell viability was determined using a Cell Counting Kit (CCK)-8 (Dojindo, Kumamoto, Japan), in which 2-(2methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt was used as a substrate (Ohuchida et al. 2004). Absorbance was measured using a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) at 450 nm (Shibasaki et al. 2012). ...
Affibody molecules specific for H-Ras and Raf-1 were evaluated for their ability to inhibit synovial cell function. Affibody molecules targeting H-Ras (Zras122, Zras220, and Zras521) or Raf-1 (Zraf322) were introduced into the MH7A synovial cell line using two delivery methods: transfection with plasmids encoding the affibody molecules or direct introduction of affibody protein using a cell-penetrating peptide reagent. Interleukin-6 (IL-6) and prostaglandin E2 (PGE2) production by MH7A cells were analyzed by enzyme-linked immunosorbent assay after stimulation with tumor necrosis factor-alpha (TNF-α). Cell proliferation was also analyzed. Phosphorylation of extracellular signal-regulated kinase (ERK) was analyzed by western blot. All affibody molecules could inhibit IL-6 and PGE2 production in TNF-α-stimulated MH7A cells. The inhibitory effect was stronger when affibody molecules were delivered as proteins via a cell-penetrating peptide reagent than when plasmid-DNA encoding the affibody moelcules was transfected into the cells. Plasmid-expressed Zras220 inhibited phosphorylation of ERK in TNF-α-stimulated MH7A cells. Protein-introduced Zraf322 inhibited the production of IL-6 and PGE2 and inhibited cell proliferation in MH7A cells. These findings suggest that affibody molecules specific for H-Ras and Raf-1 can affect intracellular signal transduction through the MAP kinase pathway to inhibit cell proliferation and production of inflammatory mediators by synovial cells.
Electronic supplementary material
The online version of this article (doi:10.1186/s13568-014-0082-3) contains supplementary material, which is available to authorized users.
Background
An affibody molecule obtained from a bioengineered staphylococcal protein was previously shown to act as an affinity binder for a wide range of targets and to develop tumour necrosis factor α (TNF-α-binding clones).
Methods
In this study, we demonstrated that affibody molecules against TNF-α could bind to recombinant TNF- on the membrane for a biochemical detection. In addition, we examined whether the affibody molecules could block binding between recombinant TNF-α and its receptor on MH7A synovial cells.
Results
When a TNF-α-binding affibody was added, the production level of inflammatory mediators IL-6 and MMP-3 in MH7A were found to decrease up to 44%. Additionally, proliferation of synovial cells was also inhibited by addition of TNF-α to the cultivation media.
Conclusion
These results suggest that affibody molecules against TNF-α could be candidate molecules for the detection of TNF- during biochemical analysis and pharmacotherapy for rheumatoid arthritis
To elucidate the pathogenesis of rheumatoid arthritis (RA), we used proteomic analysis to determine the protein profile in a synovial cell line, MH7A, established from patients with RA. Proteins were extracted from MH7A cells that were or were not stimulated with tumor necrosis factor-α (TNF-α), and then analyzed on a liquid chromatography/mass spectrometry system equipped with a unique long monolithic silica capillary. Based on the results of this proteomic analysis, we identified 2650 proteins from untreated MH7A cells and 2688 proteins from MH7A cells stimulated with TNF-α. Next, we selected 269 differentially expressed proteins that were detected only under TNF-α stimulation, and classified these proteins by performing gene ontology analysis by using DAVID as a functional annotation tool. In TNF-α-stimulated MH7A cells, we observed substantial production of plasminogen-activator inhibitor 2 and apoptosis-regulating proteins such as BH3-interacting domain death agonist, autophagy protein 5, apolipoprotein E and caspase-3. These results indicate that the upregulation of plasminogen-activator inhibitor 2 and apoptosis-regulating proteins in synovial cells in response to TNF-α stimulation might represent a predominant factor that contributes to the pathogenesis of RA.
