γ‐secretase inhibitor MRK‐003 was a gift of Merck & Co. (Whitehouse Station, NJ). 5 μM MRK‐003 was used in most experiments; exceptions are reported in figures and/or legends. Torin 1 (1‐[4‐[4‐(1‐Oxopropyl)‐1‐piperazinyl]‐3‐(trifluoromethyl) phenyl]‐9‐(3‐quinolinyl)‐benzo[h]‐1,6‐naphthyridin‐2(1H)‐one) (Tocris Bioscience, Bristol, UK) was dissolved in DMSO and used at a final concentration of 0.250 μM. Roscovitine (Cell Signaling) was dissolved in DMSO and used at a final concentration of 20 μM. UO126 (Selleck Chemicals, Huston, TX) was dissolved in DMSO, and used at a final concentration of 10 μM. Silvestrol (Medchemexpress LLC, Princeton, NJ) was dissolved in DMSO and used at a final concentration of 0.04 μM and 0.1 μM. Homoharringtonine (Tocris) was dissolved in DMSO and used at a final concentration of 0.1 μM. Insulin in solution (Sigma–Aldrich) was diluted in sterile PBS and used at the specified concentrations. pcDNA3‐RLUC‐POLIRES‐FLUC was a gift from Nahum Sonenberg (McGill University, Montreal); pCDF1‐MCS1‐EF1‐cop GFP expressing the APP C‐terminal 59 aa was a gift from Dr. Xiao Z.C. (Institute of Molecular and Cell Biology, Singapore); full length APP 695 in pCAX vector was from Addgene (Cambridge, MA). We used the empty plasmids as transfection controls.
Silvestrol
Manufactured by MedChemExpress
Sourced in United States
Silvestrol is a naturally occurring compound isolated from the plant Aglaia foveolata. It functions as a selective inhibitor of the eukaryotic translation initiation factor 4E-binding protein (4E-BP) and the mammalian target of rapamycin (mTOR) pathway. Silvestrol has been studied for its potential applications in various research fields.
Automatically generated - may contain errors
Lab products found in correlation
Roscovitine, by Cell Signaling Technology (2 mentions)
Uo126, by Selleck Chemicals (2 mentions)
Homoharringtonine, by Bio-Techne (2 mentions)
Insulin in solution, by Merck Group (2 mentions)
Full length app 695 in pcax vector, by Addgene (2 mentions)
Silvestrol, by Merck Group (2 mentions)
Zotatifin, by MedChemExpress (2 mentions)
Jnk in 8, by MedChemExpress (2 mentions)
Rocaglamide a, by MedChemExpress (2 mentions)
Jph203, by MedChemExpress (2 mentions)
Actinomycin d, by Merck Group (2 mentions)
Mda mb 468, by American Type Culture Collection (2 mentions)
U87mg, by American Type Culture Collection (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Torin1, by Bio-Techne (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Akti 1 2, by Merck Group (1 mentions)
Cymarin, by Merck Group (1 mentions)
Uo126, by Promega (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
20 protocols using silvestrol
1
Investigating Gamma-Secretase Inhibitors
2
Pharmacological Inhibition of Cellular Pathways
3
Screening of Rocaglate Derivatives
4
Silvestrol and Ribavirin Compound Storage
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Silvestrol was received from MedChem Express (Monmouth Junction, NJ, USA). Ribavirin (RBV) was received from Sigma Aldrich, St. Louis, MO, USA. All compounds were stored and diluted according to manufacturer's recommendations.
5
Establishment of CRISPR-Engineered PKR Knockout A549 Cells
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Unless specifically indicated, all chemicals were purchased from MilliporeSigma (Etobicoke, ON, Canada). PatA was a kind gift from Dr. Jerry Pelletier (McGill University, Montreal, QC, Canada), silvestrol was obtained from MedChem Express (Princeton, NJ, USA); 1 mM stocks of silvestrol and pateamine A were prepared in dimethyl sulfoxide (DMSO; MilliporeSigma, Etobicoke, ON, Canada) and stored at −80 °C. A549, Vero and Madin–Darby Canine Kidney (MDCK) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Fisher Scientific, Ottawa, ON, Canada) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Grand Island, NY, USA) and 100 U/mL penicillin + 100 µg/mL streptomycin + 20 µg/mL-glutamine (Pen/Strep/Gln; Wisent Bioproducts, St-Bruno, QC, Canada) at 37 °C in 5% CO2 atmosphere. All cell lines were purchased from the American Type Culture Collection (ATCC). PKR knock out A549 cells were generated by lentivirus-based CRISPR/Cas9 technique using guide RNA target sequence 5′-TAATACATACCGTCAGAAGC-3′ in lentiCRISPRv2 vector [21 (link)] and clonal selection.
6
Cytotoxicity Assay of Pharmacological Agents
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Human cell lines U87MG (#HTB-14), HeLa (#CCL-2), and MDA-MB-468 (HTB-132) were purchased from the American Type Culture Collection and propagated in DMEM with 10% FBS and 1% penicillin-streptomycin. Human cell line BJAB (#ACC757) was purchased from DSMZ and propagated in RPMI-1640 with 10% FBS and 1% penicillin-streptomycin. Cells were maintained at 37 °C in a 5% CO2 humidified incubator. Silvestrol (#HY-13251), rocaglamide A (#HY-19356), zotatifin (#HY-112163), JNK-IN-8 (#HY-13319), and JPH203 (#HY-100868) were purchased from MedChemExpress, actinomycin D (#A1410) was purchased from Millipore-Sigma.
7
Cellular Stress Response Induction
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For SG induction, sodium arsenite (Millipore Sigma) was added to the media to a final concentration of 500 μM and cells were returned to 37°C incubator for 50 min; Silvestrol (MedChemExpress, Monmouth Junction, NJ, USA) or Thapsigargin (Millipore Sigma) were added to the media to a final concentration of 500 nM and 1 μM, respectively, and cells were returned to 37°C incubator for 1 h. For treatment of 293A cells with translation and transcription inhibitors, cycloheximide (50 μg/ml), Actinomycin D (5 μg/ml), or Silvestrol (320 nM) were added to the media and cells were incubated for 12 h prior to lysis for western blot.
8
Optimizing Pharmacological Inhibition Assays
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Everolimus (LC Laboratories, E-4040), Torin 1 (LC Laboratories, T-7887), and silvestrol (MedChem Express, HY-13251) were reconstituted in DMSO and cells were treated at the concentrations indicated in the figures for 24 h. Phenformin (MedChem Express, HY-16397A) and eCF50632 (link) (provided by Dr. Asier Unciti-Broceta) were reconstituted in sterile water, while rotenone (Toronto Research Chemicals, R700580), oligomycin A (Sigma, 75351), CCCP (Tocris, 04-525-00), GSK621 (Adooq, A15896), PF-06409577 (Tocris, 6114), and Dasatinib (LC Laboratories, D-330) were reconstituted in DMSO. Cells were treated at the concentrations indicated in the figures for 24 h. For treatment with GSK126 (Custom synthesis, Mercachem) and EPZ6438 (MedChem Express, HY-13803) (both reconstituted in DMSO), cells were treated at a concentration of 2 µM for 7 days, with media and drug replenished on day 3 and day 6. Cells were seeded into 96-well plates for use in proliferation assays or histones were extracted for immunoblotting analysis on day 7.
9
Silvestrol Preparation Protocol
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Silvestrol was purchased from MedChem Express (Monmouth Junction, NJ) and dissolved in DMSO and stored at −80°C.
10
Silvestrol Preparation Protocol
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