Single cell suspensions of HEK293T cells were transiently transfected with 0.50 µg of the Cignal PAX6 Luciferase Reporter (QIAGEN, CCS-3042L) using FuGENE HD transfection reagent (Promega, Madison, WI) at a ratio of 3:1 (FuGENE HD: DNA) at the time of seeding. HEK293T cells were treated with buthionine sulfoximine (BSO) (Sigma-Aldrich, St. Louis, MO) to reduce intracellular GSH levels31 (link),32 (link) thusly, twenty-four h after seeding, the medium was replaced with medium containing 0 (control), 500 or 1,000 µM buthionine sulfoximine (BSO) (Sigma-Aldrich, St. Louis, MO). Cells were then incubated for a further 48 h after which luciferase reporter activity was measured using the Dual-Glo Luciferase Activity System (Promega, Madison, WI, E2920) according to the manufacturer’s instructions. Luminescence was detected using a SpectraMax M3 multimode microplate reader (Molecular Devices, San Jose, CA). The ratios of Firefly Luciferase/Renilla Luciferase luminescence were calculated for each sample. Activity in control and BSO-treated cells were compared using ANOVA with Dunnett’s correction (GraphPad Prism version 9.0, GraphPad Software, La Jolla, CA). P < 0.05 was considered significant.
Buthionine sulfoximine bso
Manufactured by Merck Group
Sourced in United States, Japan
Buthionine sulfoximine (BSO) is a synthetic compound used in research laboratories. It functions as an inhibitor of gamma-glutamylcysteine synthetase, an enzyme involved in the biosynthesis of glutathione. This inhibitory effect can be utilized to study the role of glutathione in various biological processes.
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Lab products found in correlation
N acetylcysteine nac, by Merck Group (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
N acetyl cysteine (nac), by Merck Group (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Geneprint 10 system, by Promega (2 mentions)
Auranofin, by Cayman Chemical (2 mentions)
Fv1000, by Olympus (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Spectramax m3, by Molecular Devices (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Dual glo luciferase activity system, by Promega (1 mentions)
5 5 dithiobis 2 nitrobenzoic acid dtnb, by Merck Group (1 mentions)
Triton x, by Merck Group (1 mentions)
Glutathione reductase, by Merck Group (1 mentions)
β nicotinamide adenine dinucleotide 2 phosphate reduced tetrasodium salt hydrate nadph, by Merck Group (1 mentions)
Sulfosalicylic acid, by Merck Group (1 mentions)
L glutathione reduced, by Merck Group (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Xanthine oxidase, by Merck Group (1 mentions)
34 protocols using buthionine sulfoximine bso
1
Regulation of PAX6 Expression by GSH Levels
2
Quantitative Glutathione Assay
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3
Recombinant Human BMP Bioactivity Assay
4
Cell Culture Conditions and Treatments
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The D. melanogaster embryonic cell line Kc167 was grown in Schneider medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin and streptomycin at 22°C in a humidified incubator. Primary mouse embryonic fibroblasts (MEFs) were obtained by harvesting 14.5-day C57BL/6J embryos as previously described [1 (link)]. Briefly, fetal liver and head were removed and the remainder of the embryo was mechanically disaggregated in plating medium. A suspension of single-cells was plated in DMEM (plus 10% FBS, 100U/mL penicillin and 100μg/mL streptomycin). MEFs were grown for at least three passages before experiments. MDA-MB231 and A549 cells lines were obtained from ATCC and grown in DMEM and RPMI, respectively, supplemented with 10% FBS plus antibiotics in a 37°C humidified incubator at 5% O2 and 95% CO2 atmosphere. Cells were kept and treated in the exponential phase of growing at 60–70% confluence. Buthionine sulfoximine (BSO, 2 mM) and N-acetyl-cysteine (NAC, 7.5 mM) were from Sigma-Aldrich (USA) and were pre-incubated for 8 h before MMS (40 μg/mL, IC25-50 range at 72 h for all cell lines) treatments.
5
Oxidative Stress and Inflammatory Response
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Primary LF cells were treated with H2O2 (100 μM; Wako, Tokyo, Japan), buthionine sulfoximine (BSO) (1 mM; Sigma-Aldrich, St. Louis, MO, USA), which is a glutathione synthesis suppressor that initiates OS, recombinant human TNF-α (50 ng/mL; Thermo Fisher Scientific, Waltham, MA, USA) and NAC (100 uM; Sigma-Aldrich) for 24 h. The LF cells were cultured with TNF α (50 ng/mL) and BSO (1 mM) for a duration of 30 min in association with NAC pretreatment (100 uM, 10 min) to assess the phosphorylation capacity of intracellular signaling. These experiments were conducted independently in triplicate.
6
Quantification of Total Glutathione Content
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The measurement of the total GSH content is based on the DTNB-mediated production of GS-TNB (a GSH adduct of TNB). This GS-TNB could be reduced by GR to TNB, which can be quantified as described earlier [22 (link)]. For GSH depletion, cells were treated for 6 h with 0.25 mM buthionine-sulfoximine (BSO, Sigma-Aldrich). Cells and liver samples were dissolved in 150 µL ice-cold 10 mM hydrochloric acid (HCl, Carl Roth). To lyse the cells, they were exposed to ten times ultrasound treatment (80% amplitude, 0.5 s cycle). For the liver samples, a TissueLyser (Qiagen) was used 2 × 2 min at 30 Hz for homogenization. After centrifugation for 30 s and 8000× g, 30 µL (w/v) 5% 5-sulfosalicylic acid (SSA, Sigma-Aldrich) was added to the supernatant and incubated for 10 min. A second centrifugation at 4 °C, for 15 min and 8000× g removed the denatured proteins. The extinction was measured for 5 min at 412 nm with a microplate reader (Synergy H1). All measurements were performed in triplicates using 96-well plates. The total GSH content was calculated using a standard curve and was normalized to the protein content of the samples.
7
Myeloma Cell Lines and Compound Synthesis
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Three human myeloma cell lines (JJN3, RPMI8226 and U266) were obtained from Dr. Slavica Vuckovic (QIMR Berghofer Medical Research Institute, Brisbane) and have been authenticated by the Griffith University DNA Sequencing Facility (GUDSF) using the STR profiling method (GenePrint® 10 System, Promega). Bortezomib-resistant (BR) myeloma cell lines (RPMI8226-BR and U266-BR) cells were established previously in our lab [35 ,36 (link)]. Human peripheral blood mononuclear cells (PBMCs) were collected and isolated from the whole blood of healthy individuals under the Griffith University human ethical approval number 2014/392. These cells were cultured in RPMI-1640 medium (Gibco) containing 10% (V/V) fetal bovine serum (FBS) (Bovagen), 200 mM l -glutamine with 100 U/ml penicillin and 100 ug/ml streptomycin (Invitrogen). [Au(d2pype)2]Cl was synthesized by a modification of the published procedure [37 ] using [AuCl(SMe)2] as precursor and 1,2-bis(di-2-pyridylphosphino)ethane (d2pype) obtained from Strem Chemicals Inc. Auranofin was purchased from the Cayman Chemicals (Michigan, USA), and N-acetylcysteine (NAC), sodium selenite, and buthionine sulfoximine (BSO) were purchased from Sigma Chemicals (NSW, Australia).
8
Measuring Real-time Glutathione Dynamics
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The real-time GSH dynamics in living BMDMs were determined with the fluorescent GSH probe, RealThiol RT-AM as described37 (link). Briefly, cells were plated on 2-well chamber Nunc slide pre-coated with 25 μg/ml fibrin with or without 5 μM acivicin or GGsTop. Buthionine sulfoximine (BSO, Sigma-Aldrich) was used as positive control for reducing intracellular GSH levels at 100 μM. Cells were kept at 37 °C during the entire experiment. After 24 h incubation, cells were loaded with 1 μM RT-AM probe for 5 min then fluorescence emissions after sequential excitation at 405 nm and 488 nm was acquired using sequential confocal laser scanning microscopy (Olympus FV1000; Olympus) with a 10× objective and 2× optical magnification. For each independent experiment, laser power and detector settings were set using the control/untreated cells incubated with RT-AM alone and then all settings were kept constant throughout the experiment. The ratio bound intracellular RT-AM:unbound intracellular RT-AM for each treatment condition was calculated by subtracting the 488-nm fluorescence signal from the 405 nm fluorescence signal from 30–50 cells/treatment for each independent experiment. The ratio calculated for each treatment was expressed as percentage from that of the untreated cells (control).
9
Preparation and Treatment of Cancer Cells
10
Isolation and culture of mouse hematopoietic stem cells
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