Abstract
Objective
Lipopolysaccharide (LPS) from cariogenic microorganisms and resin monomers like HEMA (2-hydroxyethyl methacrylate) included in dentin adhesive are present in a clinical situation in deep dentinal cavity preparations. Here, cell survival, expression of proteins related to redox homeostasis, and viability of mouse macrophages exposed to LPS and HEMA were analyzed with respect to the influence of oxidative stress.
Methods
Cell survival of RAW264.7 mouse macrophages was determined using a crystal violet assay, protein expression was detected by Western blotting, and HEMA- or LPS-induced apoptosis (cell viability) was analyzed by flow cytometry. Cells were exposed to HEMA (0–8 mM), LPS (0.1 μg/ml) or combinations of both substances for 24 h. The influence of mitogen-activated protein kinases (MAPK) was analyzed using the specific inhibitors PD98059 (ERK1/2), SB203580 (p38) or SP600125 (JNK), and oxidative stress was identified by the antioxidant N -acetylcysteine (NAC).
Results
Cell survival was reduced by HEMA. LPS, however, increased cell survival from 29% in cultures exposed to 8 mM HEMA, to 46% in cultures co-exposed to 8 mM HEMA/LPS. Notably, LPS-induced apoptosis was neutralized by 4–6 mM HEMA but apoptosis caused by 8 mM HEMA was counteracted by LPS. Expression of NOS (nitric oxide synthase), p47 phox and p67 phox subunits of NADPH oxidase, catalase or heme oxygenase (HO-1) was associated with HEMA- or LPS-induced apoptosis. While no influence of MAPK was detected, NAC inhibited cytotoxic effects of HEMA.
Significance
HEMA- and LPS-triggered pathways may induce apoptosis and interfere with physiological tissue responses as a result of the differential formation of oxidative stress.
1
Introduction
Initiation and progression of caries lesions are caused by a complex community of Gram-positive bacteria including mutans and non-mutans streptococci, actinomyces, lactobacilli, or bifidobacteria species accompanied by Gram-negative species like Prevotella , Porphyromonas or Fusobacterium species in a dynamic cariogenic biofilm . As caries lesions progress causing deep cavitation through the continuous degradation of dental hard tissues, these acidogenic and aciduric cariogenic microorganisms, or their surface components lipopolysaccharide (LPS) or lipoteichoic acid (LTA) released from replicating or dying cells, increasingly diffuse through dentin tubules. Both LPS, a glycolipid in the outer membrane of Gram-negative microorganisms, and LTA, a polymer linked to the membrane in Gram-positive bacteria, activate adaptive responses of immunocompetent cells including odontoblasts and macrophages of dental pulp tissues. LPS and LTA as ligands of membrane-bound Toll-like receptors (TLR) trigger signaling pathways by similar mechanisms, which finally activate regulatory molecules like MAPK (mitogen-activated protein kinases) ERK1/2 and p38/JNK or the transcription factor NF-κB .
LPS is a highly effective pro-inflammatory molecule that initiates various cell responses. While trace amounts of LPS stimulate the innate immune system via TLR4, LTA acts through a TLR2-TLR6 heterodimer leading to the NF-κB-regulated production of pro- or anti-inflammatory mediators . Noteworthy is that the same low amounts of LPS seemed to induce odontoblastic differentiation of dental pulp stem cells as a first step in dental hard tissue repair, most likely through MAPK signaling pathways . In contrast, high levels of LPS and proinflammatory cytokines may create a situation of persistent stress and chronic inflammation hindering pulp tissue repair . Moreover, LPS signaling through TLR4 was discussed as a route for triggering cell death via apoptosis, thus indicating at least a dual role for consequences resulting from of NF-κB activation . LPS-induced apoptosis in RAW264.7 mouse macrophages is a consequence of oxidative stress due to increased nitric oxide (NO) synthesis after the induction of iNOS (inducible nitric oxide synthase) expression .
It has been suggested that the thickness of dentin obviously reduces the amount of LPS that finally contacts pulp tissue. When the number of inflammatory cells observed in pulp tissues with pulpitis was compared to the caries front without a clear margin of the distribution of LPS, about two millimeters of sound dentin were considered sufficient to allow for the recovery of pulp tissues to homeostasis . Similarly, healing of the dentin-pulp complex also depends on the remaining dentin thickness when dentin adhesives and composites are used as restorative materials in dental therapy after the complete or partial removal of caries from deep carious lesions. Moderate to severe inflammatory reactions, and even progression of necrotic tissues, have been reported for adhesive systems in direct contact with pulp tissue . Likewise, inflammation in pulp tissues and adverse effects on the odontoblast cell layer were detected in non-exposed pulp tissues when the remaining dentin thickness was less than 500 μm. In addition, degeneration of pulp mesenchymal tissues and fibrous degeneration were observed in a clinical study, suggesting exposure of pulp tissues to dentin adhesives . These tissue reactions were most likely caused by monomers released from unpolymerized materials.
Diffusion of small monomers like HEMA (2-hydroxyethyl methacrylate) through dentin was repeatedly shown with remaining dentin thicknesses in the same range discussed for the penetration of LPS . Although opinions are contradictory, it is likely that monomers are available in biologically relevant concentrations sufficient to cause the adverse effects observed in pulp tissues . It has been recently shown that cells activate the expression of the redoxsensitive transcription factor Nrf2 and Nrf2-regulated enzymatic antioxidants as a consequence of monomer-induced oxidative stress . The disruption of the redox balance in cells of various origins exposed to monomers is presumably the reason for other phenomena like apoptosis or inhibition of mineralization or adequate cell responses of the innate immune system . The interaction between LPS released from cariogenic microorganisms and unpolymerized resin monomers in dentin adhesives is a particular but real clinical situation in freshly cut deep dentinal cavity preparations. Cell responses toward resin monomers could vary with pulp tissue homeostasis influenced by bacterial infection and inflammation . It has been shown that the release of pro- or anti-inflammatory cytokines from LPS-stimulated macrophages is drastically inhibited by resin monomers even after short exposure periods . As a consequence, downregulation of cytokine release by immunocompetent cells might not only hamper regulation of a local inflammation, but indirectly disturb odontoblast differentiation and reactionary dentinogenesis . In this investigation we hypothesized that resin monomers might even interfere with cellular mechanisms that support cell survival and viability in the presence of LPS. We used RAW264.7 mouse macrophages as a suitable model cell line of the innate immune system in several studies before . Here, the cells were treated with varying concentrations of the monomer HEMA in the presence of LPS. LPS derived from Escherichia coli was used as a model substance, while LPS from clinically relevant Gram-negative cariogenic microorganisms may be isolated and studied in further investigations. The role of MAPK and the influence of oxidative stress on cell survival and viability were analyzed as well.
2
Materials and methods
2.1
Chemicals and reagents
2-Hydroxyethyl methacrylate (HEMA; CAS-No. 868-779) was purchased from Merck KGaA (Darmstadt, Germany). Lipopolysaccharide (LPS; E. coli , serotype 055:B5) and N -acetylcysteine (NAC; CAS-No. 616-91-1) came from Sigma–Aldrich (Taufkirchen, Germany). PD98059, 2′-amino-3′-methoxyflavone, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole), and SP600125 (anthra[1,9-cd]pyrazole-6(2 H )-one) were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany). RPMI 1640 medium containing l -glutamine and 2.0 g/l NaHCO 3 was from PAN Biotech (Aidenbach, Germany). Fetal bovine serum (FBS), penicillin/streptomycin, and phosphate-buffered saline supplemented with 5 mM Na-EDTA (PBS-EDTA) were purchased from Life Technologies, Gibco BRL (Eggenstein, Germany). Anti-catalase (H-300, sc-50508), and anti-heme oxygenase 1 (HO-1, M-19, sc-1797) monoclonal antibodies came from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-nitric oxide synthase (NOS) (no. 2977), polyclonal antibody plus anti-rabbit IgG HRP-linked antibodies (no. 7074) were obtained from Cell Signaling (NEB Frankfurt, Germany). Goat anti-mouse IgG (H+L)-HRP conjugate was purchased from Bio-Rad Laboratories (Munich, Germany), and Amersham hyperfilm ECL came from GE Healthcare (Munich, Germany). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (clone 6C5) and CHEMICON Re-Blot Plus mild antibody stripping solution was obtained from Millipore (Schwalbach, Germany). Crystal violet came from Gibco Invitrogen (Karlsruhe, Germany), and the FACS annexin V-FITC apoptosis detection kit was obtained from R&D Systems (Minneapolis, MN, USA). All other chemicals used in the present study were at least chemical grade.
2.2
Cell culture and determination of cell survival
RAW264.7 mouse macrophages (ATCC TIB71) were maintained in RPMI 1640 medium containing l -glutamine, sodium-pyruvate and 2.0 g/l NaHCO 3 supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin following standard procedures. Cells from routine culture were seeded into 96-well plates (1 × 10 4 /well) for 24 h at 37 °C. Then, the cell cultures were exposed to 0.1 μg/ml LPS or 1, 4, 6, or 8 mM HEMA, or a combination of LPS/HEMA for 24 h. The cells were exposed in the presence or absence of 10 μM or 30 μM PD98059 (MEK1/2 inhibitor to inhibit signaling through ERK1/2), 10 μM or 30 μM SB203580 (p38 inhibitor) and 3 μM or 10 μM SP600125 (JNK inhibitor) . Cell cultures were preincubated with the inhibitors for 1 h at 37 °C. The concentrations of the inhibitors were selected after testing a wide concentration range (0–100 μM) in preliminary experiments (not shown). To analyze the influence of the antioxidant N -acetylcysteine (NAC), cell cultures were preincubated with NAC (10 mM) 1 h previous to exposure to LPS/HEMA in the presence of NAC. Exposure was stopped by discarding the exposure media. Then, the crystal violet assay was used to analyze for cell survival . Cell cultures were washed with PBS, then fixed with 1% glutaraldehyde and stained with crystal violet (0.02% in water) at room temperature. Four replicate cell cultures per experimental group were analyzed in each of four independent experiments (n = 16). Finally, optical densities were determined after dissolving the amount of crystal violet bound to the cells with 70% ethanol at 600 nm in a multi-well spectrophotometer (TECAN, Infinite 200 PRO). Cell survival (cell numbers) was calculated from optical density readings and related to untreated control cultures. Individual values of cell survival in untreated cultures were obtained from optical density readings, combined and normalized (=100%). Then, each individual value was related to 100%, and these normalized individual values were summarized as medians (plus 25–75% quartiles).
2.3
Determination of apoptosis
Cell viability and the percentage of cells in apoptosis or necrosis in cultures surviving treatment with HEMA, LPS and more compounds specified below were analyzed by flow cytometry after staining with annexin V-FITC and propidium iodide. First, RAW264.7 mouse macrophages (1.0 × 10 5 cells) were plated in each well of a 6-well plate and incubated at 37 °C for 24 h. Then, the cell cultures were exposed to 0.1 μg/ml LPS, 1, 4, 6, or 8 mM HEMA, or a combination of LPS/HEMA for 24 h. Again, cells were exposed in the presence or absence of 10 μM or 30 μM PD98059 (MEK1/2 inhibitor), 10 μM or 30 μM SB203580 (p38 inhibitor) and 3 μM or 10 μM SP600125 (JNK inhibitor). Only results obtained with 30 μM PD98059 or SB203580 or 10 μM SP600125 are presented in Section 3 for reasons of clarity. The same tendency was detected with lower concentrations (10 μM PD98059 or SB203580 or 3 μM SP600125) of the different inhibitors.
Cells were preincubated with the inhibitors for 1 h at 37 °C. In experiments testing the influence of NAC, cell cultures were preincubated with NAC (10 mM) 1 h previous to the exposure to LPS/HEMA in the presence of NAC. Exposure was stopped by discarding the exposure media, and the cells were washed with phosphate-buffered saline (PBS) at room temperature. Next, the cells were detached with PBS/5 mM EDTA, washed in PBS/5 mM EDTA, and finally collected by centrifugation. Apoptotic cell death was analyzed after staining cells with annexin V-FITC and propidium iodide (PI) as previously described . Cells were incubated in 100 μl binding buffer with annexin V-FITC and PI, and fluorescence was determined by flow cytometry (FACSCanto, Becton Dickinson, San Jose, CA, USA). FITC fluorescence (FL-1) was analyzed by a 530/30 band pass filter, and PI fluorescence (Fl-3) by a 650 nm long pass filter. Data acquisition (at least 2 × 10 4 events for each sample) was performed with FACSDiva™ 5.0.2 software, which was also used for the analysis of the number of cells in the various phases of cell death. The number of viable (annexin V-; PI-) cells was detected in the lower left quadrant (unstained) of density plots, and the percentages of cells in apoptosis (annexin V+; PI-; lower right quadrant), late apoptosis (annexin V+; PI+; upper right quadrant), and necrosis (annexin V-; PI+; upper left quadrant) were determined as well . Data from four independent experiments were collected (n = 4).
2.4
Analysis of protein expression
RAW264.7 mouse macrophages (1.5–2.0 × 10 6 cells) were cultured in cell culture plates (150 mm in diameter) at 37 °C for 24 h. The cells were exposed to HEMA (0–4–8 mM) in the presence or absence of 0.1 μg/ml LPS for 24 h as described above. Then, exposure was stopped by collecting the exposure media and floating cells. Adherent cells were washed with ice-cold PBS, detached with PBS/5 mM EDTA and collected by centrifugation. The cell pellet was resuspended and washed twice in ice-cold PBS. The cell pellet was resuspended in 1 ml PBS, then 0.5 ml buffer A (10 mM Tris HCl, 60 mM KCl, 1 mM Na 2 EDTA, 1 mM DTT, pH 7.4) was added. The cells were incubated on ice for 5 min and then collected by centrifugation in the cold. The supernatant was discarded, the cell pellet was resuspended in 1 ml buffer B (buffer A plus 0.4% NP40, 5 mM NaF, 1 mM NaVO 4 , protease inhibitor cocktail), incubated on ice for 3 min, and centrifuged for 4 min. The supernatant was collected as a cytoplasmic cell fraction. The amount of protein in this fraction was determined by a BCA protein assay (Sigma, Taufkirchen, Germany) using bovine serum albumin as a standard.
2.5
Western blot analysis
Proteins (10 μg per lane) were first separated on a 10% or 12% sodium dodecyl sulfate-polyacrylamide gel by electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane in SDS–electroblot buffer (25 mM Tris–Cl, 192 mM glycin, 20% methanol, pH 8.3) at 350 mA for 60 min. The membrane was then washed twice in TBS (25 mM Tris–Cl, 150 mM NaCl, pH 7.4) and blocked with 5% nonfat milk in TBST (TBS plus 0.1% Tween 20, pH 7.4) or bovine serum albumin (detection of GPx1/2) at room temperature for 60 min. Accordingly, the membrane was incubated overnight at cold temperatures with antibodies specific for the detection of a particular protein, as described in detail in the respective figure and legend. The membrane was then washed with TBST three times at room temperature for 10 min and primary antibodies were detected by horseradish peroxidase-conjugated secondary antibodies in TBST for 60 min. Bound secondary antibodies were visualized by enhanced chemiluminescence (ECL) after washing for 20 min in TBST and 10 min in PBS. Then, the membranes were stripped for reprobing at room temperature (15 min) using CHEMICON Re-Blot Plus mild antibody stripping solution. Finally, the membranes were washed in PBS at room temperature for 30 min, reprobed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the bound secondary antibody was visualized by ECL.
2.6
Statistical analyses
Data are presented as medians (25–75% quartiles) summarized from individual values obtained from independent experiments as described above. Differences between median values were statistically analyzed using the Mann–Whitney U test (SPSS 15.0, SPSS, Chicago, IL, USA) for pairwise comparisons among groups at the 0.05 level of significance. Median values, and the upper and lower quartiles were also plotted and shown in graphs (SigmaPlot 8.0, Systat Software, San Jose, CA, USA).
2
Materials and methods
2.1
Chemicals and reagents
2-Hydroxyethyl methacrylate (HEMA; CAS-No. 868-779) was purchased from Merck KGaA (Darmstadt, Germany). Lipopolysaccharide (LPS; E. coli , serotype 055:B5) and N -acetylcysteine (NAC; CAS-No. 616-91-1) came from Sigma–Aldrich (Taufkirchen, Germany). PD98059, 2′-amino-3′-methoxyflavone, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole), and SP600125 (anthra[1,9-cd]pyrazole-6(2 H )-one) were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany). RPMI 1640 medium containing l -glutamine and 2.0 g/l NaHCO 3 was from PAN Biotech (Aidenbach, Germany). Fetal bovine serum (FBS), penicillin/streptomycin, and phosphate-buffered saline supplemented with 5 mM Na-EDTA (PBS-EDTA) were purchased from Life Technologies, Gibco BRL (Eggenstein, Germany). Anti-catalase (H-300, sc-50508), and anti-heme oxygenase 1 (HO-1, M-19, sc-1797) monoclonal antibodies came from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-nitric oxide synthase (NOS) (no. 2977), polyclonal antibody plus anti-rabbit IgG HRP-linked antibodies (no. 7074) were obtained from Cell Signaling (NEB Frankfurt, Germany). Goat anti-mouse IgG (H+L)-HRP conjugate was purchased from Bio-Rad Laboratories (Munich, Germany), and Amersham hyperfilm ECL came from GE Healthcare (Munich, Germany). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (clone 6C5) and CHEMICON Re-Blot Plus mild antibody stripping solution was obtained from Millipore (Schwalbach, Germany). Crystal violet came from Gibco Invitrogen (Karlsruhe, Germany), and the FACS annexin V-FITC apoptosis detection kit was obtained from R&D Systems (Minneapolis, MN, USA). All other chemicals used in the present study were at least chemical grade.
2.2
Cell culture and determination of cell survival
RAW264.7 mouse macrophages (ATCC TIB71) were maintained in RPMI 1640 medium containing l -glutamine, sodium-pyruvate and 2.0 g/l NaHCO 3 supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin following standard procedures. Cells from routine culture were seeded into 96-well plates (1 × 10 4 /well) for 24 h at 37 °C. Then, the cell cultures were exposed to 0.1 μg/ml LPS or 1, 4, 6, or 8 mM HEMA, or a combination of LPS/HEMA for 24 h. The cells were exposed in the presence or absence of 10 μM or 30 μM PD98059 (MEK1/2 inhibitor to inhibit signaling through ERK1/2), 10 μM or 30 μM SB203580 (p38 inhibitor) and 3 μM or 10 μM SP600125 (JNK inhibitor) . Cell cultures were preincubated with the inhibitors for 1 h at 37 °C. The concentrations of the inhibitors were selected after testing a wide concentration range (0–100 μM) in preliminary experiments (not shown). To analyze the influence of the antioxidant N -acetylcysteine (NAC), cell cultures were preincubated with NAC (10 mM) 1 h previous to exposure to LPS/HEMA in the presence of NAC. Exposure was stopped by discarding the exposure media. Then, the crystal violet assay was used to analyze for cell survival . Cell cultures were washed with PBS, then fixed with 1% glutaraldehyde and stained with crystal violet (0.02% in water) at room temperature. Four replicate cell cultures per experimental group were analyzed in each of four independent experiments (n = 16). Finally, optical densities were determined after dissolving the amount of crystal violet bound to the cells with 70% ethanol at 600 nm in a multi-well spectrophotometer (TECAN, Infinite 200 PRO). Cell survival (cell numbers) was calculated from optical density readings and related to untreated control cultures. Individual values of cell survival in untreated cultures were obtained from optical density readings, combined and normalized (=100%). Then, each individual value was related to 100%, and these normalized individual values were summarized as medians (plus 25–75% quartiles).
2.3
Determination of apoptosis
Cell viability and the percentage of cells in apoptosis or necrosis in cultures surviving treatment with HEMA, LPS and more compounds specified below were analyzed by flow cytometry after staining with annexin V-FITC and propidium iodide. First, RAW264.7 mouse macrophages (1.0 × 10 5 cells) were plated in each well of a 6-well plate and incubated at 37 °C for 24 h. Then, the cell cultures were exposed to 0.1 μg/ml LPS, 1, 4, 6, or 8 mM HEMA, or a combination of LPS/HEMA for 24 h. Again, cells were exposed in the presence or absence of 10 μM or 30 μM PD98059 (MEK1/2 inhibitor), 10 μM or 30 μM SB203580 (p38 inhibitor) and 3 μM or 10 μM SP600125 (JNK inhibitor). Only results obtained with 30 μM PD98059 or SB203580 or 10 μM SP600125 are presented in Section 3 for reasons of clarity. The same tendency was detected with lower concentrations (10 μM PD98059 or SB203580 or 3 μM SP600125) of the different inhibitors.
Cells were preincubated with the inhibitors for 1 h at 37 °C. In experiments testing the influence of NAC, cell cultures were preincubated with NAC (10 mM) 1 h previous to the exposure to LPS/HEMA in the presence of NAC. Exposure was stopped by discarding the exposure media, and the cells were washed with phosphate-buffered saline (PBS) at room temperature. Next, the cells were detached with PBS/5 mM EDTA, washed in PBS/5 mM EDTA, and finally collected by centrifugation. Apoptotic cell death was analyzed after staining cells with annexin V-FITC and propidium iodide (PI) as previously described . Cells were incubated in 100 μl binding buffer with annexin V-FITC and PI, and fluorescence was determined by flow cytometry (FACSCanto, Becton Dickinson, San Jose, CA, USA). FITC fluorescence (FL-1) was analyzed by a 530/30 band pass filter, and PI fluorescence (Fl-3) by a 650 nm long pass filter. Data acquisition (at least 2 × 10 4 events for each sample) was performed with FACSDiva™ 5.0.2 software, which was also used for the analysis of the number of cells in the various phases of cell death. The number of viable (annexin V-; PI-) cells was detected in the lower left quadrant (unstained) of density plots, and the percentages of cells in apoptosis (annexin V+; PI-; lower right quadrant), late apoptosis (annexin V+; PI+; upper right quadrant), and necrosis (annexin V-; PI+; upper left quadrant) were determined as well . Data from four independent experiments were collected (n = 4).
2.4
Analysis of protein expression
RAW264.7 mouse macrophages (1.5–2.0 × 10 6 cells) were cultured in cell culture plates (150 mm in diameter) at 37 °C for 24 h. The cells were exposed to HEMA (0–4–8 mM) in the presence or absence of 0.1 μg/ml LPS for 24 h as described above. Then, exposure was stopped by collecting the exposure media and floating cells. Adherent cells were washed with ice-cold PBS, detached with PBS/5 mM EDTA and collected by centrifugation. The cell pellet was resuspended and washed twice in ice-cold PBS. The cell pellet was resuspended in 1 ml PBS, then 0.5 ml buffer A (10 mM Tris HCl, 60 mM KCl, 1 mM Na 2 EDTA, 1 mM DTT, pH 7.4) was added. The cells were incubated on ice for 5 min and then collected by centrifugation in the cold. The supernatant was discarded, the cell pellet was resuspended in 1 ml buffer B (buffer A plus 0.4% NP40, 5 mM NaF, 1 mM NaVO 4 , protease inhibitor cocktail), incubated on ice for 3 min, and centrifuged for 4 min. The supernatant was collected as a cytoplasmic cell fraction. The amount of protein in this fraction was determined by a BCA protein assay (Sigma, Taufkirchen, Germany) using bovine serum albumin as a standard.
2.5
Western blot analysis
Proteins (10 μg per lane) were first separated on a 10% or 12% sodium dodecyl sulfate-polyacrylamide gel by electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane in SDS–electroblot buffer (25 mM Tris–Cl, 192 mM glycin, 20% methanol, pH 8.3) at 350 mA for 60 min. The membrane was then washed twice in TBS (25 mM Tris–Cl, 150 mM NaCl, pH 7.4) and blocked with 5% nonfat milk in TBST (TBS plus 0.1% Tween 20, pH 7.4) or bovine serum albumin (detection of GPx1/2) at room temperature for 60 min. Accordingly, the membrane was incubated overnight at cold temperatures with antibodies specific for the detection of a particular protein, as described in detail in the respective figure and legend. The membrane was then washed with TBST three times at room temperature for 10 min and primary antibodies were detected by horseradish peroxidase-conjugated secondary antibodies in TBST for 60 min. Bound secondary antibodies were visualized by enhanced chemiluminescence (ECL) after washing for 20 min in TBST and 10 min in PBS. Then, the membranes were stripped for reprobing at room temperature (15 min) using CHEMICON Re-Blot Plus mild antibody stripping solution. Finally, the membranes were washed in PBS at room temperature for 30 min, reprobed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the bound secondary antibody was visualized by ECL.
2.6
Statistical analyses
Data are presented as medians (25–75% quartiles) summarized from individual values obtained from independent experiments as described above. Differences between median values were statistically analyzed using the Mann–Whitney U test (SPSS 15.0, SPSS, Chicago, IL, USA) for pairwise comparisons among groups at the 0.05 level of significance. Median values, and the upper and lower quartiles were also plotted and shown in graphs (SigmaPlot 8.0, Systat Software, San Jose, CA, USA).
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