Spore germination and outgrowth were performed in 2 × Schaeffer′s glucose (2 × SG) liquid medium (Schaeffer et al., 1965 ) supplemented with 10 mmol L−1 L‐alanine. Spores in water were first heat shocked for 30 min at 70°C, cooled on ice, and inoculated into germination medium at 37°C to obtain an initial OD600 of ~0.5. Where indicated, 0.5 mmol L−1 hydrogen peroxide (H2O2) (Sigma‐Aldrich, St. Louis MO) or 2 μmol L−1 4‐NQO (4‐Nitroquinoline‐1‐Oxide) (Sigma‐Aldrich, St. Louis MO), equivalent to a 30% lethal dose of each drug, were added to cultures after most spore germination had taken place; that is, ~15 min after the mixing of spores with germinants. The OD600 of cultures were monitored with an Ultrospec 2000 spectrophotometer (Pharmacia, Manassas Park, VA), and the values were plotted as a fraction of the initial OD600 (OD600 at time t/initial OD600) versus time. The rates of germination of disA mfd, disA uvrA, and wild‐type spores were determined in 25 mmol L−1 Tris‐HCl (pH 7.4) plus L‐alanine of spore cultures. To this end, the fall in the relative OD600 values was monitored over a period of 30 min and the linear portion employed to calculate the slope. The rate of germination of wild‐type spores was refereed as 100%.
4 nitroquinoline 1 oxide 4nqo
Manufactured by Merck Group
Sourced in United States, Germany, Brazil
4-nitroquinoline-1-oxide (4NQO) is a chemical compound used in laboratory research. It functions as a DNA-damaging agent, inducing various types of DNA lesions. The core function of 4NQO is to serve as a tool for studying DNA repair mechanisms and cellular responses to genotoxic stress. No further interpretation or extrapolation on intended use is provided.
Automatically generated - may contain errors
Lab products found in correlation
Sodium azide, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Corn oil, by MP Biomedicals (2 mentions)
Tamoxifen, by Merck Group (2 mentions)
Prolab rmh 2500, by Land O'Lakes (2 mentions)
Arecoline hydrobromide, by Tokyo Chemical Industry (2 mentions)
Mg132, by Selleck Chemicals (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
4 nqo, by Nara Biotech (2 mentions)
C57bl 6 mice, by Nara Biotech (2 mentions)
Hydrogen peroxide h2o2, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Xenolight d luciferin k salt, by PerkinElmer (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Sangon (1 mentions)
Mg132, by Merck Group (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Puromycin, by Sangon (1 mentions)
Oligonucleotides, by Takara Bio (1 mentions)
40 protocols using 4 nitroquinoline 1 oxide 4nqo
1
Bacillus subtilis Spore Germination Kinetics
2
Conditional GpNLuc/E7iresE6 Expression
3
Conditional GpNLuc/E7iresE6 Expression
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To conditionally induce GpNLuc or E7iresE6 expression, iLumiFluor or iKH and iKHP animals were intra-lingually injected using a 27-gauge needle with 0.5mg of tamoxifen (Sigma-Aldrich, cat. #: T5648) dissolved in 30µl corn oil (MP Biomedical, cat. #: 901414) either two and/or three times over a five-day period, respectively.
4NQO (4-Nitroquinoline 1-oxide; Sigma, cat. #: N8141) was dissolved in propylene glycol (Sigma, cat. #: 398039) at a concentration of 5 mg/mL and then further diluted to a final concentration of 10 or 20 µg/ml in sterile ddH2O. Following intra-lingual tamoxifen administration, animals were allowed a one-week recovery period prior to being administered 4NQO treated water for 8 weeks as previously described (Vitale-Cross et al., 2009 (link)). The 4NQO treated water was changed weekly.
4NQO (4-Nitroquinoline 1-oxide; Sigma, cat. #: N8141) was dissolved in propylene glycol (Sigma, cat. #: 398039) at a concentration of 5 mg/mL and then further diluted to a final concentration of 10 or 20 µg/ml in sterile ddH2O. Following intra-lingual tamoxifen administration, animals were allowed a one-week recovery period prior to being administered 4NQO treated water for 8 weeks as previously described (Vitale-Cross et al., 2009 (link)). The 4NQO treated water was changed weekly.
4
Evaluating DNA Damage in Neurogenic Cells Exposed to 50 Hz Magnetic Fields
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The neurogenic tumor cell lines (U251, A172 and SH-SY5Y), astrocytes and microglia were exposed or sham-exposed to 50 Hz MFs for 1, 6 or 24 h after pre-culture for 24 h. The cortical neurons were exposed or sham-exposed to 50 Hz MFs for 1, 6 or 24 h at DIV7. As positive controls, the neurogenic tumor cells (U251, A172 and SH-SY5Y) and the primary cultured neurogenic cells (astrocytes, microglia and cortical neurons) were treated with 0.1 µM of 4-Nitro-Quinoline-1-Oxide (4NQO, Sigma), a carcinogenic chemical that induces DNA damage. Immunofluorescence staining of γH2AX was carried out as previously described [30 (link)]. Primary mouse anti-γH2AX antibodies (Millipore, Temecula, CA; diluted 1:1000) were used. Alexa Fluor® 546-conjugated goat anti-mouse IgG (Zhongshan Goldenbridge Biotechnology, Beijing, China; diluted 1:300) was used as the secondary antibody. The dye 4’, 6-diamidino-2-phenylindole (DAPI, Sigma, St Louis, MO) was used to stain the cell nuclei. Over 200 cells were scored from 5 to 10 randomly selected observation fields. The number of γH2AX foci per cell and the percentage of γH2AX foci–positive cells were used as the indicators of DNA DSBs.
5
Tongue Dysplasia Mice Model Treatment
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A total of 24 female C57BL/6 mice (Nanjing Biomedical Research Institute of Nanjing University, Nanjing, China), 6‐week‐old and weighing 16 to 18 g, were divided into three groups (control, combination, and cisplatin, n = 8). Mice were fed daily with 50 μg/mL 4‐nitroquinoline 1‐oxide (4NQO, Sigma‐Aldrich, Germany) in their drinking water for 16 weeks, and then fed with distilled water for an additional 6 weeks. Fresh 4NQO or water was supplied every week. At week 18, mice with visible lesions of tongue dysplasia were treated with different agents, solvent as negative control, metformin (100 mg/kg, intraperitoneal injection) and 4SC‐202（80 mg/kg, intragastric administration), cisplatin (1 mg/kg, intraperitoneal injection) as positive control for 4 weeks. All animals were euthanized on week 22, and tissue retrieval was done as described previously. All animals were monitored daily for general behavioral abnormalities, signs of toxicity, illness, or discomfort.
All the animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Sun Yat‐sen University.
All the animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Sun Yat‐sen University.
6
Studying OTUD6B and β-TrCP Interactions
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OTUD6B, OTUD6BC188S, β‐TrCP and SNAIL were inserted into the pLVX‐IRES vector. The OTUD6B and β‐TrCP truncated mutants were cloned into the pEBG‐GST vector. RARα was inserted into pENTER vector. The DUBs (including ATXN3, BRCC3, COPS5, USP15, USP47, UCHL1, OTUB1, OTUD6B, and VCPIP1) were cloned into the pcDNA3 vector. The OTUD6B shRNA, β‐TrCP, and SNAIL shRNA sequences were cloned into the pSIH‐H1 vector. The siRNAs targeting RARα were purchased from RiboBio (Guangzhou, China). All primers used for plasmid construction and the target sequences of shRNAs and siRNAs are shown in Table S1 , Supporting Information.
The reagents were as follows: ATRA (Sigma, USA), MG132 (Sigma, USA), CHX (Sigma, USA), cisplatin (Sigma, USA), 4‐nitroquinoline 1‐oxide (4NQO, Sigma, USA), XenoLight D‐Luciferin K+ salt (PerkinElmer, USA), TRIzol (Invitrogen, USA), puromycin (Sangon Biotech, China), G418 (Sigma, USA), 4,6‐diamidino‐2‐phenylindole (DAPI, Sangon Biotech, China).
The information on antibodies used in this study is provided in TableS2 , Supporting Information.
The reagents were as follows: ATRA (Sigma, USA), MG132 (Sigma, USA), CHX (Sigma, USA), cisplatin (Sigma, USA), 4‐nitroquinoline 1‐oxide (4NQO, Sigma, USA), XenoLight D‐Luciferin K+ salt (PerkinElmer, USA), TRIzol (Invitrogen, USA), puromycin (Sangon Biotech, China), G418 (Sigma, USA), 4,6‐diamidino‐2‐phenylindole (DAPI, Sangon Biotech, China).
The information on antibodies used in this study is provided in Table
7
Recombinant Protein Expression in E. coli and H. volcanii
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The oligonucleotides used in this study were purchased from Takara (Dalian, China). Escherichia coli strain DH5α was used for gene cloning, and the Rosetta2(DE3)pLysS strain was used for recombinant protein expression. The expression vectors pET28a and pDEST17 were used to express recombinant proteins. The H. volcanii strain and plasmids for gene knockout were provided by Dr Allers (University of Nottingham, UK). Pyrococcus furiosus genomic DNA was purchased from the American Type Culture Collection (ATCC). KOD-plus DNA polymerase was purchased from Toyobo. Nickel–nitrilotriacetic acid resin was purchased from Bio-Rad. The crosslinking reagent BS3 and the DNA damage reagents 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), and methyl methanesulfonate (MMS), methyl methanesulfonate (MMS), N3-methyladenine and adenine were purchased from Sigma–Aldrich (St Louis, MO). All other chemicals and reagents were of analytical grade.
8
HNSCC Cell Line and Tissue Protocols
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All experimental reagents including 4-nitroquinoline 1-oxide (4-NQO), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cisplatin (CDDP), and 5-fluorouracil (5-FU) were purchased from Sigma. The HNSCC cell lines, clinical human HNSCC tissues, and animal procedure were obtained and described elsewhere (60 (link), 61 (link)).
9
Oral Tumorigenesis in C57BL/6J Mice
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Six-week-old male C57BL/6JNarl mice (n=150) were purchased from the National Laboratory Animal Center. The mice were handled in accordance with the Animal Care and Use Guidelines of the China Medical University, and the study protocol was approved by the Institutional Animal Care Use Committee. Mice were randomized assigned into one of nine groups. The experiments were controlled as previously described 27 (link). The carcinogens, 200 μg/ml 4-nitroquinoline 1-oxide (4-NQO; Sigma-Aldrich, St. Louis, MO, USA) and 500 μg/ml arecoline hydrobromide (Tokyo Chemical Industry Co. LTD, Tokyo, Japan), were used to induce oral tumorigenesis in mice. The experimental diets were prepared by mixing fenofibrate (0.3%, w/w) with the powdered chow diets (Prolab® RMH 2500 PMI Nutrition International, LLC, MO, USA). At the end of 28 weeks, all mice were euthanized. The tongues were then excised and prepared for pathological, immunohistochemical, and Western blot analysis.
10
Induction of Oral Carcinogenesis in Mice
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The induction of oral carcinogenesis was performed as previously described [29 (link), 33 (link)]. Briefly, 4-nitroquinoline-1-oxide (4NQO) (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in ethylene glycol (Sigma-Aldrich) at 50 µg/mL or 200 µg/mL and was stored at 4°C. Experimental mice received 4NQO daily for 28 weeks, and the control mice received drinking water only. After 28 weeks, the mice were euthanized and the tongue, cervical lymph nodes, liver, lungs, stomach, duodenum, jejunum, ileum and large intestine were collected for microscopic analysis.
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