Phosphatidylcholine (Phospholipon 90 G® - PL90G) (93.0 ± 3.0% phosphatidylcholine) was obtained from Nottermann Phospholipid GMBH, Köln, Germany, and was used without any purification. Absolute ethanol, sodium cholate hydrate (SC) (minimum purity of 99%), Trypan Bleu dye solution (0.4% v/v), a 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide (MTT) dye test (TLC purity ≥97.5%), sodium dimethyl sulfoxide (DMSO), phosphate buffer (PBS) solution, and DL-Sulforaphane (SFN) were purchased from Sigma-Aldrich (Milan, Italy). Eagle’s Minimum Essential Medium (EMEM) culture, fetal bovine serum (FBS 10×), penicillin (100 UI/mL)-streptomycin (100 μg/mL) solution (1% v/v), and Trypsin/EDTA (1×) solution were obtained from GIBCO (Invitrogen Corporation, Giuliano Milanese (Mi), Italy). Spectra/Por cellulose membranes (Cut off 10,000), used for drug release tests, were provided by Spectrum Laboratories Inc. SK-MEL-28, human melanoma cells, were provided by the Istituto Zooprofilattico of Modena and Reggio Emilia, Italy. Double distilled pyrogen-free water was used throughout experimental investigations. All other materials and solvents used in this study were of analytical grade.
Dl sulforaphane sfn
Manufactured by Merck Group
Sourced in United States
DL-Sulforaphane (SFN) is a synthetic organic compound. It is a naturally occurring isothiocyanate found in cruciferous vegetables such as broccoli. SFN acts as an antioxidant and has been studied for its potential health benefits.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Absolute ethanol, by Merck Group (1 mentions)
Sodium cholate hydrate, by Merck Group (1 mentions)
Mmp 9 antibody, by Cell Signaling Technology (1 mentions)
Mmp 2 antibody, by Santa Cruz Biotechnology (1 mentions)
Transwell plates for invasion assay, by BD (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc apoptosis assay kit, by Keygen Biotech (1 mentions)
Mts assay kit, by Promega (1 mentions)
Sbi 0206965, by Merck Group (1 mentions)
Anti bach1 sc 271211, by Santa Cruz Biotechnology (1 mentions)
Anti gfp antibody sc 9996, by Santa Cruz Biotechnology (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Cell Signaling Technology (1 mentions)
Mg132, by Cell Signaling Technology (1 mentions)
Prl cmv plasmid, by Promega (1 mentions)
Dual luciferase system protocol, by Promega (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
6 protocols using dl sulforaphane sfn
1
Phosphatidylcholine-based Nanocarrier Formulation
2
Molecular Mechanisms of Sulforaphane-Induced Apoptosis
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Culture media were purchased from Hyclone (Logan, Utah, USA), FBS and penicillin–streptomycin were acquired from Invitrogen (Carlsbad, CA, USA). MMP-9 antibody was purchased from Cell Signaling Technology, Inc (Shanghai, China). MMP-2 antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Bax, Bad, Bcl-2, cytochrome C and α-tubulin were purchased from Proteintech Group, Inc (Chicago, USA). d , l -Sulforaphane (SFN) was acquired from Sigma (St Louis, MO, USA). MTS assay kit was purchased from Promega (Madison, USA). Transwell plates for invasion assay were bought from BD Biosciences (Bedford, MA, USA). BCA protein assay kit was obtained from Thermo Scientific Pierce Protein Research Products (Rockford, IL., USA). Annexin V-FITC Apoptosis Assay Kit was acquired from Nanjing KeyGEN Biotech (Nanjing, China).
3
Sulforaphane-mediated Nrf2 Activation
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DL -Sulforaphane (Sfn) (#S4441), MG132 (#474787), SBI0206965 (#SML1540-5MG), Actinomycin D (#A1410), Hemin (#H9039) were from Sigma-Aldrich (Vienna, Austria) and dissolved in DMSO (to at least 1000X stocks), stored at −80°C and added to the cells at the indicated concentrations (maximal final DMSO concentration of 0.2%). Antibodies against AMPKα (#2532), Keap1 (#7705), Nrf2 (#12721), α/β-Tubulin (#2148), Lamin (#12586) as well as secondary horseradish peroxidase-coupled antibodies against mouse (#7076) and rabbit (#7074) were purchased from Cell Signaling (Frankfurt am Main, Germany) and used at 1:1,000 dilution for Western Blotting and 1:100 for immunoprecipitation. Anti-Bach1 (#sc-271211) and anti-GFP antibody (#sc-9996) came from Santa Cruz (Heidelberg, Germany), used at 1:300 dilutions for Western Blotting and 1:50 for immunoprecipitation. The α-actin antibody (#69100) was from MP Biologicals (Illkirch, France) and used in a 1:5,000 dilution.
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4
Validating NRF2 Antibody Specificity
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In order to judge the NRF2 antibody specificity and sensitivity, NRF2 p.Thr80Lys mutant and wild-type cells were treated separately with the proteasome inhibitor MG-132 (10 mM in DMSO, Cell Signaling, Cambridge, UK) and d ,l -Sulforaphane SFN (10 mM in DMSO, Sigma, Saint Louis, MO, USA) as recommended. DMSO was used as a control. Cells were treated with 10 µM MG-132 or 10 µM d ,l -Sulforaphane for the last 16 h before collecting. Stabilization of NRF2 was analyzed by immunoblotting. The specificity of the used NRF2 antibody was confirmed as advised in the literature42 (link) (Supplementary Fig. 3 ).
5
Investigating Promoter Activity in RAW-264.7 Cells
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To investigate the promoter activity, RAW-264.7 cells were plated in 24-well polystyrene plates (1 × 105 cells per well) and transfected with 1μg of reporter plasmids using LIPOFECTAMINE 2000 reagent (Invitrogen, Carlsbad, CA, USA). The following plasmids were employed in the assays: 3xARE and Nrf2-WT. Luciferase activity was normalized using 40 ng of pRL-CMV plasmid (Promega Corp., Madison, WI, USA), 24 h after transfection the cells were treated. After treatment, cells rested for 24 h and were washed with PBS and lysed according to the Dual Luciferase System protocol (Promega Corp.), and analyzed using the GloMax®;-Multi detection system (Promega Corp., Madison, WI, USA). Positive controls consisting of cells stimulated with 10 mM DL-sulforaphane (SFN) (Sigma-Aldrich) were used to induce the activation of ARE and Nrf-2 gene expression.
6
Sulforaphane Cytotoxicity on Osteosarcoma Cells
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All cell culture reagents were purchased from Life Technologies (Carlsbad, CA-USA), unless otherwise stated. Human osteosarcoma MG-63 cell line (ATCC, Manassas, VA-USA) was cultured in α-Minimum Essential Medium supplemented with 10% foetal bovine serum, 2.5 μg/ml fungizone, and 100 U/ml penicillin-100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. When ∼80% cell confluence was reached, cells were trypsinised with Trypsin-EDTA (0.25% Trypsin, 1 mM EDTA) and subcultured at a split ratio of 1∶10. D,L-sulforaphane (SFN; Sigma-Aldrich, St. Louis, MO-USA) was dissolved in DMSO (Sigma-Aldrich, St. Louis, MO-USA) at a 10 mM stock concentration and stored at −20°C. Cells were allowed to adhere for 24 h and medium was replaced with fresh medium containing 0, 5, 10, and 20 μM SFN. Cells were exposed for 24 and 48 h.
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