Highlights
- •
In ions were released from the Ag–Pd–Au–In alloy.
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In ions resulted in cytotoxicity to oral keratinocytes.
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Intracellular reactive oxygen species increased due to In ions.
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In ions induced the terminal differentiation of oral keratinocytes.
Abstract
Objective
Dental alloys containing indium (In) have been used in dental restoration for two decades; however, no study has investigated the biological effects of In ions, which may be released in the oral cavity, on human oral keratinocytes. The objective of the present study was to investigate the biological effects of In ions on human oral keratinocyte after confirming their release from a silver–palladium–gold–indium (Ag–Pd–Au–In) dental alloy.
Methods
As a corrosion assay, a static immersion tests were performed by detecting the released ions in the corrosion solution from the Ag–Pd–Au–In dental alloy using inductively coupled plasma atomic emission spectroscopy. The cytotoxicity and biological effects of In ions were then studied with In compounds in three human oral keratinocyte cell lines: immortalized human oral keratinocyte (IHOK), HSC-2, and SCC-15.
Results
Higher concentrations of In and Cu ions were detected in Ag–Pd–Au–In ( P < 0.05) than in Ag–Pd–Au, and AgCl deposition occurred on the surface of Ag–Pd–Au–In after a 7-day corrosion test due to its low corrosion resistance. At high concentrations, In ions induced cytotoxicity; however, at low concentrations (∼0.8 In 3+ mM), terminal differentiation was observed in human oral keratinocytes. Intracellular ROS was revealed to be a key component of In-induced terminal differentiation.
Significance
In ions were released from dental alloys containing In, and high concentrations of In ions resulted in cytotoxicity, whereas low concentrations induced the terminal differentiation of human oral keratinocytes via increased intracellular ROS. Therefore, dental alloys containing In must be biologically evaluated for their safe use.
1
Introduction
High-carat gold (Au) alloys have been used for centuries in dental restorations; however, the market price of Au has rapidly increased with continued use . The traditional high-carat Au alloys have been substituted with low-carat Au alloys, such as the Ag–Cu–Au alloy . Since a gold-yellow color dental alloy with two white colored metals, i.e., palladium (Pd) and indium (In), was invented two decades ago, the silver–palladium–gold–indium (Ag–Pd–Au–In) dental alloy has been widely used in dental restoration. Three primary reasons for the use of this alloy are its (1) gold-yellow color, (2) increased bonding strength with porcelain, and (3) increased tarnish and corrosion resistance when containing 5–10% In . However, the corrosion resistance of dental alloys containing In depends on the proportion of each element; thus, In ions could potentially be released into the oral cavity from the corroded alloy, and this potential release of In ions has been underestimated to date .
The embryotoxicity and teratogenicity of In ions has been reported in the literature . In addition, In itself exhibits cytotoxicity in human leukocytes and lymphocytes . Nevertheless, In has been used as a supplemental element in gallium-based liquid alloys instead of dental amalgam due to the relative biocompatibility with L929 (mouse fibroblasts) for <1 mM of In ions . A biological study of extracts from dental alloy containing In was performed with Balb/c 3T3 (mouse embryos) and exhibited toxicity; in addition, the released In ions concentration was determined to be approximately 0.2 mM for 72 h of incubation in tissue culture media . However, cytotoxicity tests of In ions with human oral keratinocytes have not been performed despite the use of dental alloys containing In for dental restoration. Previous studies have demonstrated that the cytotoxicity results could differ dramatically based on the cell lines employed, especially those originating from different types of tissue . Therefore, cytotoxicity studies of dental materials with human oral keratinocytes have been widely performed because extracts or ions released from dental materials anatomically encounter oral keratinocytes of epithelium and thus affect the biocompatibility of the oral epithelium . Hence, this study considers In-induced cytotoxicity with human oral keratinocytes.
Aside from cytotoxicity, other biological effects of metal ions have been studied in the use of dental alloys in dental restoration. In clinical situations, the released metal ions might be diluted to non-cytotoxic levels in the oral cavity by saliva. Hence, the biological effect of metal ions under non-cytotoxic levels must be investigated such that these ions can be safely used as components of dental alloys . Generally, a high concentration of metal ions induces cytotoxicity; however, other biological results are observed at lower concentrations . The metal ions Au 3+ , Ag + , Pd 2+ , Cu 2+ , Ni 2+ , and In 3+ resulted in no cytotoxicity at concentrations of up to 5 mM . A few millimoles of Ni 2+ or Cu 2+ , which is under the cytotoxic concentration, have been demonstrated to affect epithelial–mesenchymal transition (EMT) and fibrosis in human skin keratinocytes, which are considered to be associated with cancerization . Therefore, the biological effects of In at non-cytotoxicity concentrations should be evaluated for the use of In in dental alloys.
The biological effects of metal ions on oral keratinocytes under cytotoxic concentrations can be divided into two different types: cancerization and terminal differentiation of keratinocytes. EMT is used as a representative event to evaluate cancerization. During EMT, fibronectin is up-regulated, and over-expression of epidermal growth factor receptor (EGFR) phosphorylation is detected . Fibronectin is a mesenchymal marker that is highly expressed in mesenchymal cells and is up-regulated in cancerization . EGFR is the transmembrane receptor in keratinocytes for uptaking various growth factors (i.e., epithermal growth factor, tissue growth factor-alpha) and plays a pivotal role in regulating keratinocyte proliferation, differentiation, and transformation . The over-expression of EGFR was detected in oral keratinocyte cancerization . However, the terminal differentiation of oral keratinocyte, which plays a pivotal role in maintaining the physical barrier of oral mucosa, could be induced. During terminal differentiation, keratinocytes produce increasingly more keratin and involucrin, which contributes to the physical barrier and formation of a cell envelop to protect the outermost part of the oral epithelium .
Biological events induced by metal ions have been determined to be related to an increased concentration of intracellular reactive oxygen species (ROS) because these species serve as signaling intermediates in cellular signaling pathways, including cell differentiation, proliferation and apoptosis . Previous studies suggested that increased intracellular ROS may play an early causal role in the terminal differentiation in keratinocytes . Therefore, the terminal differentiation of oral keratinocytes via increased intracellular ROS might be considered a potential biological event .
Thus, the objective of the present study was to investigate the biological effects of In ions on human oral keratinocytes after confirming the release of In ions from a Ag–Pd–Au–In dental alloy. The null hypothesis of this study was that In ions do not induce significant biological effects on human oral keratinocytes.
2
Materials and methods
2.1
Dental alloys
Two dental alloy compositions, Ag–Pd–Au (70% Ag–15% Pd–5% Au) and Ag–Pd–Au–In (60% Ag–15% Pd–10% In–5% Au), were selected for investigation after a preliminary study, and rectangular-shaped specimens were prepared using a conventional metal alloy casting procedure. To precisely control the composition and compare the characteristics of the alloys, we fabricated our own specimens rather than purchasing commercial alloys. Briefly, each alloy was prepared by melting Ag, Pd, Au, and/or In pellets (purity 99.99%, LS nikko, Ulsan, Korea). After weighing each component for the designated composition, three samples of 15 g of the mixture were melted three times in an arc melter to promote chemical homogeneity. The chamber was evacuated to 5 × 10 −3 Torr, and high-purity argon gas was introduced until the pressure reached 200 torr before melting. After cooling to room temperature (RT), the ingots were cut, and pieces were used to make rectangular specimens (34 mm × 13 mm) with a thickness of 1.5 mm using a conventional lost wax casting technique, resulting in a total surface area of 10.25 cm 2 . Each specimen was prepared following the sample treatment methods in ISO 10271 . Briefly, the specimen was blasted with 125 μm of pure alumina and mirror-polished using SiC sandpaper with up to # 2000 grit and a woolen-cotton disk on the polisher (Polisher & Grinder, ECOMET III, Buehler, Lake Bluff, IL, USA). The specimen was cleaned ultrasonically in ethanol (99.9%, Duksan Pure Chemical, Kyunggi, Korea) and deionized water (DW) for 15 min and then dried in a vacuum chamber at 23 °C.
2.2
Static immersion tests
Static immersion tests were performed according to ISO 10271, which provides corrosion media to determine the anti-corrosion properties of all metallic materials used in dentistry . The corrosion solution was composed of 0.1 M lactic acid (C 3 H 6 O 3 ) and 0.1 M sodium chloride (NaCl) in distilled water; the pH was adjusted to 2.3. Each specimen was incubated with 1 mL of the corrosion solution per cm 2 for the specified period (1, 4 or 7 days). Inductively coupled plasma atomic emission spectroscopy (ICP-OES, 7300DV, Perkin Elmer, Waltham, MA, USA) was used to determine the ion concentration (Ag, Pd, Au, Cu, In). After a digital pH meter (Orion 4 Star, Thermo Fisher Scientific Inc., Singapore) calibrated to pH 4.01 and pH 7.00 immediately before use was used to measure the change of pH in the incubated corrosion solution , ICP-OES was performed as described previously using the corrosion solution collected after the set incubation time . The entire solution was changed for each measurement, and 1 mL of fresh corrosion solution was added after washing the specimen three times with DW and air brushing. Each concentration of each ion at each point accumulated after each measurement in the same alloy specimen following incubation. The total concentration was divided by the surface area, and the final concentration was determined. A field-emission scanning electron microscopy (FE-SEM, JSM-7001F, JEOL, Japan) was performed before and after the corrosion test. The metal specimen was immersed in the 1 mL of corrosion solution at 37 °C. The pH of the corrosion solution was measured after 1, 4, and 7 days of incubation. The experiments were performed in triplicate.
2.3
X-ray diffraction
Polished dental alloy specimens were evaluated by high resolution X-ray diffraction (HR-XRD, Rigaku, Kuraray, Japan) using a Cu-Kα radiation source of 40 kV and 40 mA between 2 θ values of 20° and 80°. Each specimen was measured in the same manner after the corrosion test. XRD analysis software (MDI JADE 9, v 9.3.3, Materials Data Inc., Livermore, CA, USA) and the International Center for Diffraction Data (ICDD)-PDF database were used to identify all the phases in the samples.
2.4
Cell culture
Three different human keratinocyte lines were used to confirm the general biological effect (not the cell-type-specific effect) in this study; these lines were immortalized human oral keratinocytes (IHOK) and two types of oral squamous cell carcinoma (OSCC). IHOK were provided by the Department of Oral Pathology and Oral Cancer Research Institute at Yonsei University College of Dentistry (Seoul, Korea) . HSC-2 (JCRB0622, Japanese Collection of Research Bioresources, Japan) and SCC-15 (CRL-1623, American Type Culture Collection, USA) cells were purchased and used as representative epithelial-like and spindle-like OSCC keratinocytes, respectively . IHOK and HSC-2 were incubated in a DMEM/F-12 3:1 mixture media (Welgene, Daegu, Korea), and SCC-15 was incubated in a DMEM/F-12 1:1 mixture media (Welgene). A total of 1 × 10 4 cells were grown for 24 h in each well of a 96-well plate (SPL, Gyeongido, Korea) and cultured in the abovementioned culture medium with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Invitrogen, Grand Island, NY, USA) at 37 °C in a fully humidified atmosphere of 5% CO 2 . Cell assays were performed after 24 h of incubation with serially diluted InCl 3 or In 2 (SO 4 ) 3 . All the powder was purchased from Sigma–Aldrich (USA).
2.5
Cytotoxicity tests
A cytotoxicity test was performed according to ISO 10993-12 . Dilutions of InCl 3 or In 2 (SO 4 ) 3 solution, matching 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05 and 0 mM of In ions, in phosphate buffered saline (PBS) were incubated with adherent cells for 24 h, and cell viability was measured using a water-soluble tetrazolium (WST) salt assay (Ez-Cytox, Daeillab, Seoul, Korea). Control cells were exposed to appropriate fresh culture medium for 24 h. Five wells in a 96-well plate were used for each condition. The cell viability results for each test group were expressed as the ratio of the optical density value for each test sample to each control sample following the WST assay. The optical density was read using a microplate absorbance reader (Epoch, BioTek, Winooski, VT, USA) at 450 nm. The cytotoxicity results were also confirmed using a live and dead cell imaging kit (Molecular Probes, Eugene, OR, USA) for observation under a fluorescence inverted microscope (Eclipse TE2000-U, Nickon, Japan). The 494/517 or 517/617 nm (excitation/emission) wavelengths were used to detect living and dead cells, which exhibited green and red fluorescence, respectively. Serially diluted In ion concentrations were added to the cell culture media (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 mM) to detect the pH in the cell culture media exposed to InCl 3 or In 2 (SO 4 ) 3 . All the experiments were performed in triplicate.
2.6
Detection of intracellular ROS
The cells were pretreated with or without 4 mM n-acetyl cysteine (NAC) for 1 h and then stained with 1 mM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) for 1 h. After the cells were treated with InCl 3 for 24 h and washed twice with PBS, reactive fluorescent units (RFUs, excitation 480 nm, emission 530 nm) were detected with a fluorescence microplate reader (Varioskan flash, Thermo Scientific, Waltham, MA, USA). The results were analyzed by normalizing the absorbance (intracellular ROS level) values to the cell number, which adjusts for cell plating differences among the wells. Five wells in a 96-well plate were used for each condition, and three independent experiments were performed.
2.7
Fibronectin and EGFR expression
Ten thousand cells were seeded in each 96-well plate for 24 h in each cell line. After each well was treated with 0, 0.05, 0.2, 0.8 and 3.2 mM of In ions for 24 h, the expression of fibronectin (ab108847, Abcam, Cambridge, MA) and phosphorylation of EGFR (#62205, Thermo Scientific, Waltham, MA, USA) were determined using an in vitro enzyme-linked immunosorbent assay (ELISA) kit according to each manufacturer’s protocol. Briefly, a standard or sample was added to each well and incubated at RT. After washing twice with PBS and adding the appropriate primary detection antibody, a streptavidin–peroxidase conjugate and chromogen substrate were added to detect fibronectin, and a horseradish peroxidase conjugated reagent and 3,3′,5,5′-tetramethylbenzidine were added to detect phosphorylated EGFR. The absorbance was detected at 450 nm after adding the stop solution. Five wells were used for each condition, and the experiments were performed in triplicate.
2.8
Detection of keratin
After being seeded onto 96-well plates for 24 h and treated with InCl 3 for another 24 h, three types of cells were independently fixed with 100% methanol for 15 min at −20 °C and incubated with 5% bovine serum albumin (Sigma–Aldrich, USA) at RT for 60 min. The cells were incubated with Alexa Fluor 488 conjugated pan-keratin (c11) mouse monoclonal antibody (Cell Signaling Technology, #4523, Danvers, MA, USA) at 4 °C overnight to stain keratin 4, 5, 6, 8, 10, 13, and 18 from human cells with green fluorescence. This step was followed by incubation with DAPI (#90229, Millipore, Billerica, MA, USA) to detect the nucleus as blue fluorescence after washing with PBS. A fluorescence microscope (EVOS, Life Technology, Grand Island, NY, USA) was used to detect both keratin and the nucleus. Keratin expression was analyzed using Image J2x (NIH, USA) and Photoshop CS3 (Adobe, USA) software. The density of green fluorescence in each image was measured after conversion of the images into 8-bit gray scale. The density of keratin expression was plotted after being normalized to the cell number, as counted by DAPI staining. Therefore, the keratin expression per cell number was determined in each image. All experiments were performed four times.
2.9
Detection of involucrin
Western blotting was performed to study the terminal differentiation of oral keratinocytes induced by In ions. IHOK was treated with various concentrations of InCl 3 , whereas the NAC group was pretreated with NAC (4 mM) for 1 h before the aforementioned treatment. After 3 days of incubation, cells were collected for the preparation of whole-cell lysate using ice-cold cell lysis buffer (#9803, Cell Signaling Technology) supplemented with a protease inhibitor cocktail (Halt protease inhibitor, Thermo Scientific, USA) and 1 mM PMSF. The cells were completely re-suspended in extraction buffer and kept in ice for 30 min with occasional mixing, and cell lysates were collected after spinning at 14,000 × g for 10 min at 4 °C. The protein concentrations were measured using a BCA protein assay kit (Pierce, Thermo Scientific, USA). The obtained whole-cell lysate was resolved in sodium dodecyl sulfate-polyacrylamide gel and transferred onto a nitrocellulose transfer membrane (Whatman, Germany) using a semi-dry transfer cell (Trans-blot SD, Bio-rad, CA, USA). The transferred membranes were blocked in 5% non-fat milk (prepared in Tris-buffered saline supplemented with 0.1% Tween-20; TBST) at ambient temperature for 1 h and incubated with primary antibody at 4 °C overnight. Involucrin (#SC-28557, Santa Cruz Biotechnology, TX, USA) and beta-actin (#4970, Cell Signaling Technology) were used as the primary antibody to detect the terminal differentiation protein and housekeeping protein, respectively. The membranes were washed with TBST for 1 h every 10 min before being incubated with HRP-coupled secondary antibody (#7074, Cell Signaling Technology) for 2 h at ambient temperature. After the membranes were washed with TBST for 2 h every 10 min, protein signals were visualized using enhanced chemiluminescence (Lumi pico solution, Dogen, Korea) and exposed to X-ray film (AGFA, Belgian). Scanning densitometry was performed to normalize the involucrin to beta-actin expression, and the density of the blotted band was calculated using Image J2x after conversion of the scanned images into 8-bit gray scale. The experiments were performed in triplicate.
2.10
Statistical analysis
Statistical analyses for comparison of two different conditions were performed using an independent t -test. For comparison over three different conditions, one-way analysis of variance (ANOVA) and Tukey’s method were adapted as a post hoc test. The significance was set at P = 0.02 and P < 0.05. All the experiments were run in at least triplicate. SPSS PASW 18.0 (SPSS Inc., Chicago, IL, USA) was used for all the statistical analyses.
2
Materials and methods
2.1
Dental alloys
Two dental alloy compositions, Ag–Pd–Au (70% Ag–15% Pd–5% Au) and Ag–Pd–Au–In (60% Ag–15% Pd–10% In–5% Au), were selected for investigation after a preliminary study, and rectangular-shaped specimens were prepared using a conventional metal alloy casting procedure. To precisely control the composition and compare the characteristics of the alloys, we fabricated our own specimens rather than purchasing commercial alloys. Briefly, each alloy was prepared by melting Ag, Pd, Au, and/or In pellets (purity 99.99%, LS nikko, Ulsan, Korea). After weighing each component for the designated composition, three samples of 15 g of the mixture were melted three times in an arc melter to promote chemical homogeneity. The chamber was evacuated to 5 × 10 −3 Torr, and high-purity argon gas was introduced until the pressure reached 200 torr before melting. After cooling to room temperature (RT), the ingots were cut, and pieces were used to make rectangular specimens (34 mm × 13 mm) with a thickness of 1.5 mm using a conventional lost wax casting technique, resulting in a total surface area of 10.25 cm 2 . Each specimen was prepared following the sample treatment methods in ISO 10271 . Briefly, the specimen was blasted with 125 μm of pure alumina and mirror-polished using SiC sandpaper with up to # 2000 grit and a woolen-cotton disk on the polisher (Polisher & Grinder, ECOMET III, Buehler, Lake Bluff, IL, USA). The specimen was cleaned ultrasonically in ethanol (99.9%, Duksan Pure Chemical, Kyunggi, Korea) and deionized water (DW) for 15 min and then dried in a vacuum chamber at 23 °C.
2.2
Static immersion tests
Static immersion tests were performed according to ISO 10271, which provides corrosion media to determine the anti-corrosion properties of all metallic materials used in dentistry . The corrosion solution was composed of 0.1 M lactic acid (C 3 H 6 O 3 ) and 0.1 M sodium chloride (NaCl) in distilled water; the pH was adjusted to 2.3. Each specimen was incubated with 1 mL of the corrosion solution per cm 2 for the specified period (1, 4 or 7 days). Inductively coupled plasma atomic emission spectroscopy (ICP-OES, 7300DV, Perkin Elmer, Waltham, MA, USA) was used to determine the ion concentration (Ag, Pd, Au, Cu, In). After a digital pH meter (Orion 4 Star, Thermo Fisher Scientific Inc., Singapore) calibrated to pH 4.01 and pH 7.00 immediately before use was used to measure the change of pH in the incubated corrosion solution , ICP-OES was performed as described previously using the corrosion solution collected after the set incubation time . The entire solution was changed for each measurement, and 1 mL of fresh corrosion solution was added after washing the specimen three times with DW and air brushing. Each concentration of each ion at each point accumulated after each measurement in the same alloy specimen following incubation. The total concentration was divided by the surface area, and the final concentration was determined. A field-emission scanning electron microscopy (FE-SEM, JSM-7001F, JEOL, Japan) was performed before and after the corrosion test. The metal specimen was immersed in the 1 mL of corrosion solution at 37 °C. The pH of the corrosion solution was measured after 1, 4, and 7 days of incubation. The experiments were performed in triplicate.
2.3
X-ray diffraction
Polished dental alloy specimens were evaluated by high resolution X-ray diffraction (HR-XRD, Rigaku, Kuraray, Japan) using a Cu-Kα radiation source of 40 kV and 40 mA between 2 θ values of 20° and 80°. Each specimen was measured in the same manner after the corrosion test. XRD analysis software (MDI JADE 9, v 9.3.3, Materials Data Inc., Livermore, CA, USA) and the International Center for Diffraction Data (ICDD)-PDF database were used to identify all the phases in the samples.
2.4
Cell culture
Three different human keratinocyte lines were used to confirm the general biological effect (not the cell-type-specific effect) in this study; these lines were immortalized human oral keratinocytes (IHOK) and two types of oral squamous cell carcinoma (OSCC). IHOK were provided by the Department of Oral Pathology and Oral Cancer Research Institute at Yonsei University College of Dentistry (Seoul, Korea) . HSC-2 (JCRB0622, Japanese Collection of Research Bioresources, Japan) and SCC-15 (CRL-1623, American Type Culture Collection, USA) cells were purchased and used as representative epithelial-like and spindle-like OSCC keratinocytes, respectively . IHOK and HSC-2 were incubated in a DMEM/F-12 3:1 mixture media (Welgene, Daegu, Korea), and SCC-15 was incubated in a DMEM/F-12 1:1 mixture media (Welgene). A total of 1 × 10 4 cells were grown for 24 h in each well of a 96-well plate (SPL, Gyeongido, Korea) and cultured in the abovementioned culture medium with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Invitrogen, Grand Island, NY, USA) at 37 °C in a fully humidified atmosphere of 5% CO 2 . Cell assays were performed after 24 h of incubation with serially diluted InCl 3 or In 2 (SO 4 ) 3 . All the powder was purchased from Sigma–Aldrich (USA).
2.5
Cytotoxicity tests
A cytotoxicity test was performed according to ISO 10993-12 . Dilutions of InCl 3 or In 2 (SO 4 ) 3 solution, matching 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05 and 0 mM of In ions, in phosphate buffered saline (PBS) were incubated with adherent cells for 24 h, and cell viability was measured using a water-soluble tetrazolium (WST) salt assay (Ez-Cytox, Daeillab, Seoul, Korea). Control cells were exposed to appropriate fresh culture medium for 24 h. Five wells in a 96-well plate were used for each condition. The cell viability results for each test group were expressed as the ratio of the optical density value for each test sample to each control sample following the WST assay. The optical density was read using a microplate absorbance reader (Epoch, BioTek, Winooski, VT, USA) at 450 nm. The cytotoxicity results were also confirmed using a live and dead cell imaging kit (Molecular Probes, Eugene, OR, USA) for observation under a fluorescence inverted microscope (Eclipse TE2000-U, Nickon, Japan). The 494/517 or 517/617 nm (excitation/emission) wavelengths were used to detect living and dead cells, which exhibited green and red fluorescence, respectively. Serially diluted In ion concentrations were added to the cell culture media (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 mM) to detect the pH in the cell culture media exposed to InCl 3 or In 2 (SO 4 ) 3 . All the experiments were performed in triplicate.
2.6
Detection of intracellular ROS
The cells were pretreated with or without 4 mM n-acetyl cysteine (NAC) for 1 h and then stained with 1 mM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) for 1 h. After the cells were treated with InCl 3 for 24 h and washed twice with PBS, reactive fluorescent units (RFUs, excitation 480 nm, emission 530 nm) were detected with a fluorescence microplate reader (Varioskan flash, Thermo Scientific, Waltham, MA, USA). The results were analyzed by normalizing the absorbance (intracellular ROS level) values to the cell number, which adjusts for cell plating differences among the wells. Five wells in a 96-well plate were used for each condition, and three independent experiments were performed.
2.7
Fibronectin and EGFR expression
Ten thousand cells were seeded in each 96-well plate for 24 h in each cell line. After each well was treated with 0, 0.05, 0.2, 0.8 and 3.2 mM of In ions for 24 h, the expression of fibronectin (ab108847, Abcam, Cambridge, MA) and phosphorylation of EGFR (#62205, Thermo Scientific, Waltham, MA, USA) were determined using an in vitro enzyme-linked immunosorbent assay (ELISA) kit according to each manufacturer’s protocol. Briefly, a standard or sample was added to each well and incubated at RT. After washing twice with PBS and adding the appropriate primary detection antibody, a streptavidin–peroxidase conjugate and chromogen substrate were added to detect fibronectin, and a horseradish peroxidase conjugated reagent and 3,3′,5,5′-tetramethylbenzidine were added to detect phosphorylated EGFR. The absorbance was detected at 450 nm after adding the stop solution. Five wells were used for each condition, and the experiments were performed in triplicate.
2.8
Detection of keratin
After being seeded onto 96-well plates for 24 h and treated with InCl 3 for another 24 h, three types of cells were independently fixed with 100% methanol for 15 min at −20 °C and incubated with 5% bovine serum albumin (Sigma–Aldrich, USA) at RT for 60 min. The cells were incubated with Alexa Fluor 488 conjugated pan-keratin (c11) mouse monoclonal antibody (Cell Signaling Technology, #4523, Danvers, MA, USA) at 4 °C overnight to stain keratin 4, 5, 6, 8, 10, 13, and 18 from human cells with green fluorescence. This step was followed by incubation with DAPI (#90229, Millipore, Billerica, MA, USA) to detect the nucleus as blue fluorescence after washing with PBS. A fluorescence microscope (EVOS, Life Technology, Grand Island, NY, USA) was used to detect both keratin and the nucleus. Keratin expression was analyzed using Image J2x (NIH, USA) and Photoshop CS3 (Adobe, USA) software. The density of green fluorescence in each image was measured after conversion of the images into 8-bit gray scale. The density of keratin expression was plotted after being normalized to the cell number, as counted by DAPI staining. Therefore, the keratin expression per cell number was determined in each image. All experiments were performed four times.
2.9
Detection of involucrin
Western blotting was performed to study the terminal differentiation of oral keratinocytes induced by In ions. IHOK was treated with various concentrations of InCl 3 , whereas the NAC group was pretreated with NAC (4 mM) for 1 h before the aforementioned treatment. After 3 days of incubation, cells were collected for the preparation of whole-cell lysate using ice-cold cell lysis buffer (#9803, Cell Signaling Technology) supplemented with a protease inhibitor cocktail (Halt protease inhibitor, Thermo Scientific, USA) and 1 mM PMSF. The cells were completely re-suspended in extraction buffer and kept in ice for 30 min with occasional mixing, and cell lysates were collected after spinning at 14,000 × g for 10 min at 4 °C. The protein concentrations were measured using a BCA protein assay kit (Pierce, Thermo Scientific, USA). The obtained whole-cell lysate was resolved in sodium dodecyl sulfate-polyacrylamide gel and transferred onto a nitrocellulose transfer membrane (Whatman, Germany) using a semi-dry transfer cell (Trans-blot SD, Bio-rad, CA, USA). The transferred membranes were blocked in 5% non-fat milk (prepared in Tris-buffered saline supplemented with 0.1% Tween-20; TBST) at ambient temperature for 1 h and incubated with primary antibody at 4 °C overnight. Involucrin (#SC-28557, Santa Cruz Biotechnology, TX, USA) and beta-actin (#4970, Cell Signaling Technology) were used as the primary antibody to detect the terminal differentiation protein and housekeeping protein, respectively. The membranes were washed with TBST for 1 h every 10 min before being incubated with HRP-coupled secondary antibody (#7074, Cell Signaling Technology) for 2 h at ambient temperature. After the membranes were washed with TBST for 2 h every 10 min, protein signals were visualized using enhanced chemiluminescence (Lumi pico solution, Dogen, Korea) and exposed to X-ray film (AGFA, Belgian). Scanning densitometry was performed to normalize the involucrin to beta-actin expression, and the density of the blotted band was calculated using Image J2x after conversion of the scanned images into 8-bit gray scale. The experiments were performed in triplicate.
2.10
Statistical analysis
Statistical analyses for comparison of two different conditions were performed using an independent t -test. For comparison over three different conditions, one-way analysis of variance (ANOVA) and Tukey’s method were adapted as a post hoc test. The significance was set at P = 0.02 and P < 0.05. All the experiments were run in at least triplicate. SPSS PASW 18.0 (SPSS Inc., Chicago, IL, USA) was used for all the statistical analyses.
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