For the treatment of melanoma cells with inhibitors, 2×105 melanoma cells per well were seeded in six-well plates. After 4 h, the cells were treated with 20 mM of the PI3 kinase inhibitor LY-294002, 20 mM of Wortmannin (both Sigma-Aldrich, St Louis, MO, USA) or a control substance for 16 h. For the inhibition of XPO1, cells were incubated with 10 ng/ml of leptomycin B (LMB, Sigma-Aldrich) for 20 h. For MEK inhibition, cells were treated with 10 μM PD98059 or 10 μM U0126 (Merck Millipore, Billerica, MA, USA) for 48 h. RNA polymerase was inhibited using 5 μM α-amanitin (AppliChem, Darmstadt, Germany). Treatment with MG132 (Merck Millipore) at final concentrations of 5, 10 and 20 μM for 24 hours was used to inhibit the proteasome. For protein kinase inhibition, cells were treated with 100 μM staurosporine (Merck Millipore) for 16 hours.
α amanitin
Manufactured by AppliChem
Sourced in Germany
α-amanitin is a chemical compound that functions as a toxin. It is found naturally in certain species of poisonous mushrooms. This compound acts by inhibiting RNA polymerase II, which is essential for transcription of genetic information in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Ly294002, by Merck Group (2 mentions)
Wortmannin, by Merck Group (2 mentions)
Mg132, by Merck Group (1 mentions)
Pd98059, by Merck Group (1 mentions)
Staurosporine, by Merck Group (1 mentions)
U0126, by Merck Group (1 mentions)
Leptomycin b lmb, by Merck Group (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mg132, by Cayman Chemical (1 mentions)
Kt5720, by Merck Group (1 mentions)
G 6983, by Santa Cruz Biotechnology (1 mentions)
4 protocols using α amanitin
1
Melanoma Cell Inhibitor Treatments
2
Nuclei Isolation and Nuclear Run-on Assay
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Nuclei of AX2 cells were isolated by sucrose gradient centrifugation as described before 60 (link) with modification in cell lysis. Here, 5 x 108 cells were resuspended in 20 ml lysis buffer (50 mM Tris pH 7, 5 mM MgCl2, 20 mM KCl, 10 % sucrose, 1 mM dithiothreitol, 0.2 mM phenylmethylsulphonyl chloride) and disrupted mechanically by the use of a cell homogenizer (Isobiotec, Heidelberg) with a clearance of 10 µm.
Nuclear run on reactions were carried out as described previously.61 (link) For inhibition of RNA Polymerase II, α-Amanitin (Applichem) was added to a concentration of 330 ng/ml for 15 minutes or 30 minutes, before addition of [32P]UTP (Hartmann). RNA was extracted with 1 ml Trizol® (Life technologies)/0.2 ml chloroform. The aqueous phase was filtered through Sephadex G50 and RNA was denatured at 95°C prior to slot blot hybridization. For the slot blot, 100 pmol of each DNA oligonucleotide probe (Sigma Aldrich) were transferred to nitrocellulose filters by using a vacuum slot blot apparatus. Oligonucleotides were then chemically crosslinked as described62 (link) and hybridized with radioactively labeled run-on RNA at 42°C over night. After three washing steps with 0,1%SDS/0,1xSSC, filters were exposed to Phosphoimager plates for 24 h.
Nuclear run on reactions were carried out as described previously.61 (link) For inhibition of RNA Polymerase II, α-Amanitin (Applichem) was added to a concentration of 330 ng/ml for 15 minutes or 30 minutes, before addition of [32P]UTP (Hartmann). RNA was extracted with 1 ml Trizol® (Life technologies)/0.2 ml chloroform. The aqueous phase was filtered through Sephadex G50 and RNA was denatured at 95°C prior to slot blot hybridization. For the slot blot, 100 pmol of each DNA oligonucleotide probe (Sigma Aldrich) were transferred to nitrocellulose filters by using a vacuum slot blot apparatus. Oligonucleotides were then chemically crosslinked as described62 (link) and hybridized with radioactively labeled run-on RNA at 42°C over night. After three washing steps with 0,1%SDS/0,1xSSC, filters were exposed to Phosphoimager plates for 24 h.
3
Cell Culture and Reagent Protocols
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Human HuH-7 and A549 cells, African green monkey Vero 76 cells, and CV-1 cells were maintained in Dulbecco modified Eagle medium (Gibco, catalog no. 21969035) supplemented with 10% fetal calf serum, 2 mM l -glutamine (Gibco, catalog no. 25030081), and 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco, catalog no. 15140122). The specific use of the respective cell line is indicated in the figure legends. The RPB1-specific inhibitor α-amanitin was purchased from AppliChem (A1485,0001), the nuclear export inhibitor leptomycin B was purchased from US Biological (L1671-38B), and the proteasomal inhibitor MG132 was purchased from Cayman Chemical.
4
Melanoma Cell Line Inhibition Assay
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The melanoma cell line Mel Ei (150,000 cells) were cultured in six-well plates and were treated for 24 h with different inhibitors (SB431542 InvivoGen 2 μM; S3I-201 Sigma-Aldrich 20 mM; Dorsomorphin Tebi-bio 2 mM; LY-294002 Sigma-Aldrich 20 mM; Wortmannin Sigma-Aldrich 10 mM; UO126 Calbiochem 10 mM; Vemurafenib Active Biochem 100 μM; KT5720 Merck Millipore 100 nM; Bisindolylmaleimide II Santa Cruz 10 μM; Gö 6983 Santa Cruz 10 μM; Wnt Agonist Calbiochem 10 μM). For the stability assay the cells were treated with α-Amanitin (AppliChem, 5 mM). After isolation of total RNA, quantitative RT-PCR was performed to detect relative gene expression. Normalization was based on mRNA input in this experiment.
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