To study the development of oSCC including stages of early and late dysplasia, mice ingested the carcinogen 4-nitroquinoline-1-oxide (4NQO; 100 μg/ml; Sigma Aldrich, St. Louis, MO) with their drinking water on an unrestricted basis for 16 weeks. Water was changed weekly with freshly prepared 4NQO dissolved in propylene glycol (5 mg/ml) [66 (link)]. Control mice received water containing the equivalent dilution of propylene glycol alone. Functional allodynia, as a result of progressive 4NQO-induced carcinogenesis, was assessed using the dolognawmeter once a week for the full duration of the model (28 weeks). At the end of the experiment, tongue tissue was harvested, fixed in 10% buffered formalin, and processed for paraffin embedding and slide preparation. Tissue sections were cut at 4 μm and stained with hematoxylin & eosin (H & E) by the New York University Langone Medical Center Histopathology Core. A blinded oral pathologist at the New York University Bluestone Center for Clinical Research performed histopathological analyses to score dysplastic grade or determine the presence of oSCC.
4 nitroquinoline 1 oxide
Manufactured by Merck Group
Sourced in United States
4-nitroquinoline-1-oxide is a laboratory reagent used in various scientific and research applications. It is a heterocyclic organic compound with a defined chemical structure and properties. The core function of this product is to serve as a tool in experimental and analytical procedures, without specific interpretation or extrapolation on its intended use.
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Lab products found in correlation
2 nitrofluorene, by Merck Group (4 mentions)
2 aminoanthracene, by Merck Group (4 mentions)
Sodium azide, by Merck Group (3 mentions)
Mitomycin c, by Merck Group (3 mentions)
9 aminoacridine, by Merck Group (2 mentions)
Potassium chloride (kcl), by Merck Group (2 mentions)
Methyl methanesulfonate, by Merck Group (2 mentions)
Biotin, by Merck Group (2 mentions)
Aphidicolin, by Merck Group (2 mentions)
Camptothecin, by Merck Group (2 mentions)
3 methylcholanthrene, by Merck Group (2 mentions)
Absolute methanol, by Merck Group (1 mentions)
Isoflurane, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Magnesium chloride, by Merck Group (1 mentions)
Bleomycin, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Sodium arsenite, by Merck Group (1 mentions)
Yeast peptone dextrose, by Formedium (1 mentions)
Ccm0105, by Formedium (1 mentions)
17 protocols using 4 nitroquinoline 1 oxide
1
4NQO-Induced Oral Squamous Cell Carcinoma in Mice
2
Mutagenicity and Genotoxicity Assays
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Sodium carboxymethyl cellulose (CMC-Na), dimethyl sulfoxide (DMSO), nicotine-adenine dinucleotide phosphate (NADP), glucose-6-phosphate (G6P), sodium azide, 2-nitrofluorene, 9-aminoacridine, 4-nitroquinoline-1-oxide, 2-aminoanthracene, fetal bovine serum (FBS), absolute methanol, cyclophosphamide monohydrate (CPA), sodium thiopentone barbiturate, isoflurane, magnesium chloride, potassium chloride, and sodium phosphate salts were obtained from Sigma-Aldrich (St. Louis, MO). Salmonella typhimurium TA98, TA100, TA1535, and TA1537 strains were sourced from the National Collection of Type Cultures (London, UK). Escherichia coli WP2 uvrA/pKM101 was obtained from the National Collection of Industrial, Food, and Marine Bacteria (Scotland, UK). Lyophilized rat liver S9 fraction and Bacto Agar were purchased from Celsis International (Cambridge, UK) and Becton Dickinson, Sparks, MD, respectively. The clinical chemistry and hematology reagents were obtained from Instrumentation Laboratory India Pvt. Ltd. (New Delhi, India) and Siemens (Munich, Germany), respectively.
3
Yeast Strains and Plasmids for Cellular Studies
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The yeast WT strains and the isogenic histone mutants used in this work (Supplementary Table S1 ) were from the SHIMA library, kindly provided by Dr. Ali Shilatifard (Kansas, USA)24 (link). All other strains used in this study (Supplementary Table S1 ) were the WT BY4741 and the indicated isogenic mutants derived from the nonessential haploid mutant (D.R. laboratory resource) and the TAP-tagged collections provided by Dr. Hugo Wurtele (Montreal, Canada). Epitope-tagging of strains at the endogenous gene locus was performed as previously described25 (link). Cells were grown at 30 °C for 24 h in either Yeast Peptone Dextrose (YPD, FORMEDIUM CCM0105) or SD minimal media. The single-copy plasmid pSTE5-GFP carrying the entire STE5 gene under its promoter and tagged with GFP was kindly provided by Dr. Peter M. Pryciak (University of Massachusetts medical school, Worcester, MA, USA). The plasmid pSTE5-Myc was constructed by gap repair using pTW438 as the backbone and as previously decsribed26 (link). All chemical reagents including rapamycin, methyl methanesulfonate, 4-nitroquinoline-1-oxide, bleomycin and sodium arsenite were purchased from Sigma, St Louis, USA.
4
Monitoring Oral Premalignant Lesions in Mice
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Starting from 2 months of age, mice were administered drinking water containing 50 μg/ml 4-nitroquinoline 1-oxide (4NQO; Sigma-Aldrich, St. Louis, MO, USA) [25 (link)]. Premalignant oral lesions developed on the tongue and their appearance was monitored endoscopically using a 1.9mm x 30° endoscope and a 1088 HD camera (Stryker, Kalamazoo, MI, USA). Once lesions developed at approximately 6–7 weeks of 4NQO treatment, the 4NQO treatment was terminated (Fig 3 ). Progression of premalignant oral lesions was monitored in groups of 20 mice each on a weekly basis by sedating mice with inhaled isoflurane (Piramal Healthcare, Bethlehem, PA, USA) and examining the oral cavities by endoscopy. Endoscopic images were used for quantitating lesion severity in a blinded manner. Severity was scored between 1 and 4 based on the number of visible lesions, the area of the lesions relative to the area of the tongue, ulceration, thickness, and gross pathology [26 (link)]. The parameters for scoring are outlines in Table 1 . Scores of 1 to 4 were given for each of the 4 categories, added, and then divided by 4. In prior studies, this scoring method of the overall appearance of the lesions coincided with histologic analysis of paraffin-embedded sections of tongue tissue by an oral pathologist [27 (link)].
5
Bacterial Mutagenicity Assay Protocols
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The following chemicals—2-aminoanthracene (2-AA), benzo[a]pyrene (B[a]P), sodium azide (SA), 2-nitrofluorene (2-NF), 4-nitroquinoline-1-oxide (4NQO), acridine mutagen ICR 191 (ICR-191), cyclophosphamide monohydrate (CPA), acridine orange solution (AO), ethyl methanesulfonate (EMS), dimethylsulfoxide (DMSO), potassium chloride (KCl), magnesium sulfate, citric acid monohydrate, potassium phosphate dibasic anhydrous, sodium ammonium phosphate, glucose, sodium chloride, tryptophan, histidine, biotin, methyl alcohol, and glacial acetic acid—were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Oxoid Nutrient Broth No. 2 and Bacto agar were purchased from Thermo Scientific Inc. (Waltham, MA, USA) and BD (Franklin Lakes, NJ, USA), respectively. S9 mix (5% for bacterial reverse mutation assay and 30% for chromosomal aberration assay, v/v) was prepared using Aroclor 1254-induced rat liver S9 (Molecular Toxicology Inc., Boone, NC, USA) supplemented with cofactor-I (8 μM MgCl2, 33 μM KCl, 5 μM glucose-6-phosphate, 4 μM nicotinamide adenine dinucleotide phosphate, 4 μM nicotinamide adenine dinucleotide, 100 μM sodium phosphate buffer, pH 7.4; Wako Pure Chem. Ind., Osaka, Japan). S9 mix was prepared fresh prior to use and kept on ice during the experiment. Information on chemicals and reagents not described here is supplied in the relevant sections in “Materials and methods” section.
6
Yeast Stress Response Assay Protocol
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All routine chemicals used in the study were acquired from Himedia, Merck, or SRL Pvt. Ltd. Mumbai, India. Drugs such as cycloheximide (CHX), 4-nitroquinoline 1-oxide (NQO), fluconazole (FLC), anisomycin (ANI), cerulenin (CER), and fluorescent dye Nile red (NR) were procured from Sigma-Aldrich Co., St. Louis, MO, USA. Reagents such as ammonium acetate, Polyethylene Glycol (PEG), Lithium Acetate (LiAc), sodium chloride (NaCl), Tris-HCl, EDTA, and dimethyl sulfoxide (DMSO) were also purchased from Sigma-Aldrich Sigma-Aldrich Co., St. Louis, MO, USA. Oligonucleotides were obtained from Sigma-Aldrich, Bangalore, India and are listed in the Table S1 .
7
Preparation of AZOX and 4NQO Solutions
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AZOX was purchased from Sigma-Aldrich (MO, United States) and dissolved in dimethyl sulfoxide (DMSO) to form the stock solution of 10 mg/ml. For cell experiments, the solution was diluted with a cell culture medium to different concentrations of AZOX solutions. The final concentration of DMSO in the medium was <0.2% and had a minor effect on cell growth. While for animal experiments, the solution was diluted with corn oil to prepare high-dose (25 mg/kg) and low-dose (5 mg/kg) AZOX solutions. 4-nitro-quinoline-1-oxide (4NQO) was obtained from Sigma-Aldrich (MO, United States) and dissolved in 1,2-propanediol to form the stock solution of 5 mg/ml. This stock solution was then diluted to a concentration of 50 μg/ml with purified water.
8
DNA Damage Response Assay in Cell Lines
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HEK 293T, Rad18−/− HCT116 (Shiomi et al., 2007 (link)), and U2OS cells were grown in high glucose DMEM (Gibco) with penicillin/streptomycin (Gibco), glutamine (Gibco), and 10% fetal bovine serum. Cells were damaged with MMS (Sigma-Aldrich), mitomycin C (Sigma-Aldrich), EMS (Sigma-Aldrich), 4-nitroquinoline 1-oxide (Sigma-Aldrich), aphidicolin (Sigma-Aldrich), camptothecin (Sigma-Aldrich), actinomycin D (EMD Millipore), etoposide (Sigma-Aldrich), hydrogen peroxide (Sigma-Aldrich), or cis-platinum (Sigma-Aldrich) at the indicated concentrations and times. Cells were also treated with ATR inhibitor (ATR-45; Charrier et al., 2011 (link)), ATM inhibitor (KU55933; Abcam), MG132 (Sigma-Aldrich), or LLnL (EMD Millipore) at the indicated concentrations and times.
9
Comprehensive Biochemical Reagents Protocol
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Itraconazole (ITC), Clotrimazole (CTR), Ketoconazole (KTZ), Miconazole (MCZ) and Voriconazole (VOR), Anisomycin (ANI), Rhodamine 6G (R6G), 4-nitroquinoline 1-oxide (NQO), Adenosine triphosphate (ATP), Oligomycin (OM), DL-Dithiothreitol (DTT), Sorbitol, Phenylmethanesulfonyl fluoride (PMSF), p-Tosyl-L-lysine chloromethyl ketone (TLCK), and Tosyl phenylalanyl chloromethyl ketone (TPCK) were procured from Sigma Chemical Co. (St. Louis, MO). Protease inhibitors leupeptin, aprotinin, Pepstatin were obtained from G-biosciences, MO, USA). Fluconazole (FLC) was a generous gift from Ranbaxy Laboratories, India. Oligonucleotides were procured from Sigma Genosys, India and are listed in Table S2 . Anti-GFP monoclonal antibody was purchased from Santa Cruz Biotechnology Inc. (Texas, USA). All other routine chemicals were purchased from Fisher-scientific, Mumbai, India.
10
Mutagenicity Evaluation of Chemicals
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The Salmonella typhimurium strains (TA97a, TA98, TA100, TA102, TA1535, and TA1537), Escherichia coli strain (WP2uvrA), and lyophilized rat liver S9 fraction were purchased from Molecular Toxicology Inc., Boone, NC. Mouse lymphoma cells (L5178Y, clone-3.7.2c TK+/−), DMEM, and fetal bovine serum (FBS) were obtained from ATCC, Manassas, VA; Bacto agar was procured from Becton Dickinson (Sparks, Maryland). 2-Aminoanthracene, 2-nitrofluorene, sodium azide, 9-aminoacridine, 4-nitroquinoline-1-oxide, mitomycin C, cyclophosphamide, and colchicine were purchased from Sigma-Aldrich Corporation (St. Luis, MO). The clinical chemistry and hematology reagents were sourced from ILab Aries (Milano, Italy) and Mindray (Shenzhen, China). The analytical and laboratory reagents were purchased from Sigma-Aldrich Chemicals (Bengaluru, India).
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