A375 (CRL-1619) and Mewo (HTB-65) melanoma cells were purchased at American Type Culture Collection (Manassas, VA, USA) and normal human epidermal melanocytes at PromoCell (Heidelberg, Germany). SK-Mel-2, SK-Mel-5 and SK-Mel-28 melanoma cells were kindly provided by Dr Laura Poliseno (CRL-ITT, Pisa, Italy). Cell lines were authenticated by short tandem repeat profiling. Human melanoma samples (Supplementary Table S1 ) were obtained after approved protocols by the local Ethics Committee, and processed as previously described.7 (link), 8 (link) Patient-derived SSM2c, M26c, M33x melanoma cells were already described8 (link) and M51 cells were derived from a subcutaneous melanoma metastasis. Lentiviruses were produced in HEK-293T cells as already described.58 (link) Cells were periodically tested for Mycoplasma contamination by 4’,6-diamidino-2-phenylindole inspection and PCR. CI-1040 and SCH772984 (Sigma, St Louis, MO, USA) were used for 16 h, respectively, at 1 μM and 0.5 μM , and transduced cells were selected with puromycin (2 μg/ml) (Sigma).
Sch772984
Manufactured by Merck Group
Sourced in United States
SCH772984 is a small-molecule inhibitor developed by Merck Group. It functions as a selective inhibitor of the extracellular-signal-regulated kinase (ERK) pathway.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin, by Merck Group (2 mentions)
Sch772984, by Selleck Chemicals (2 mentions)
Plx4720, by Selleck Chemicals (2 mentions)
Gsk269962a, by Axon Medchem (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Bafilomycin a1, by Merck Group (2 mentions)
Doxycycline, by Merck Group (2 mentions)
Oligomycin a, by Merck Group (2 mentions)
Rotenone, by Merck Group (2 mentions)
Antimycin, by Merck Group (2 mentions)
Chloroquine diphosphate, by Merck Group (2 mentions)
Ci 1040, by Merck Group (1 mentions)
A375 crl 1619, by American Type Culture Collection (1 mentions)
Rpmi 1640 medium, by Sartorius (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
H1299, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle s medium dmem, by Sartorius (1 mentions)
H1299, by Sartorius (1 mentions)
Mk2206 2hcl, by Merck Group (1 mentions)
Mk 2206 2hcl, by Selleck Chemicals (1 mentions)
12 protocols using sch772984
1
Melanoma Cell Line Authentication and Drug Treatment
2
Lung Cancer Cell Line Cultivation and Inhibitor Treatment
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The human LUAD cell lines A549 and H1299 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and authenticated by short tandem repeat (STR) profiling. The HEK293T cell line was acquired from the Integrated Hospital of Traditional Chinese Medicine (TCM-Integrated Hospital) of Southern Medical University (Guangzhou, P.R. China). These cell lines were analyzed for mycoplasma and were verified to be mycoplasma-free. A549 and H1299 cells were cultured in RPMI 1640 medium (Biological Industries, Bet Haemek, Israel) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA). HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Biological Industries) supplemented with 10% FBS. All cells were incubated in a humidified incubator at 37°C with 5% CO2. The AKT1 inhibitor MK-2206 2HCl and the MAPK3/1 inhibitor SCH772984 were purchased from Selleck Chemicals (Houston, TX, USA). For inhibitor treatment, 5 mmol/L MK-2206 2HCl and/or 5 mmol/L SCH772984, or dimethyl sulfoxide (DMSO; Sigma-Aldrich) alone for control, were freshly added to the cell culture every 24 h.
3
Cell Culture Conditions and Viral Infections
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CRFK cells [European Collection of Cell Cultures (ECACC)] were grown in minimum essential medium (MEM, Gibco #31095-029) containing L-glutamine (Gibco) and 1% nonessential amino acids (Gibco), and supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin, in a 5% CO2 environment. U2OS and A549 cells were grown in DMEM (Dulbecco's Modified Eagle's Medium, Gibco #41966), supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), glutamine and 1% penicillin-streptomycin (Gibco) in a 5% CO2 chamber. GFP-G3BP1 expressing, G3BP1/2 double knockout U2OS, UBAP2L knockout HeLa, MEFs S51A and RNase L knockout A549 cells have been described elsewhere (Brocard et al., 2020 (link); Burke et al., 2020 (link); Cirillo et al., 2020 (link)). Arsenate, SCH772984, puromycin and cycloheximide were all purchased from Sigma. All inhibitors were added to the cell culture media and incubated at 37°C for the indicated times. For viral infections, the FCV strain Urbana was used as previously described at a multiplicity of infection (MOI) equal to 1, and at 0.2 MOI for VFS generation (Humoud et al., 2016 (link)). After 1 h the inoculation was removed, and the cells were incubated with fresh medium for 5 h post infection (h.p.i.).
4
Evaluating ERK Inhibitor in RCC Cells
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The human renal epithelial cell line HK-2 and seven human RCC cell lines (786-O, CaKi-1, Caki-2, A-704, 769-P, A498, and ACHN) were obtained from the American Type Tissue Collection. The cells were grown in Eagle's Minimal Essential Media supplemented with 10% fetal bovine serum (FBS; Hyclone), and the cells were incubated at 37°C and in 5% CO 2 . We purchased SCH772984 (an ERK-selective inhibitor) from Sigma.
5
Lentiviral shRNA and Dox-Inducible MEK/BRAF Constructs
6
Inhibition of Kinase Pathways
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Inhibitors MEK inhibitor GSK1120212/Trametinib, ROCK inhibitor Fasudil, BRAF inhibitor PLX-4720 were bought from Selleck Chemicals, Houston, TX, USA. ERK inhibitor SCH772984 was provided by Merck & Co, Whitehouse Station, NJ, USA (via a MTA). ROCK inhibitor GSK269962A was from Axon Medchem, Groningen, the Netherlands. Metabolic poison phenyl arsine oxide (PAO) and vehicle dimethylsulfoxide (DMSO) were from Sigma-Aldrich, St. Louis, MO, USA.
7
SCH772984 Inhibitor Preparation
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The ERK1/2-selective inhibitor SCH772984 was provided by A. Samatar (Merck). Inhibitors for in vitro studies were dissolved in dimethyl sulfoxide (DMSO) to yield a 10 mM or 20 mM stock concentration and stored at −20 or −80 °C, respectively.
8
Colon Cancer Cell Line Cultivation
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The SW480, SW620, Caco-2, DLD-1, HCT116, and LoVo human colon cancer cell lines were obtained from the General Surgery Laboratory of Peking University First Hospital (Beijing, China). These cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% foetal bovine serum (FBS; both from Gibco; Thermo Fisher Scientific, Inc.) at 37°C and supplemented with 5% CO2. hEGF (cat. no. E9644), which was used as an ERK1/2 activator in our research (15 (link)), was purchased from Sigma-Aldrich; Merck KGaA. SCH-772984, a selective inhibitor of Erk1/2 (hereinafter called EI), dissolved in dimethylsulfoxide (DMSO) was purchased from Selleckchem. Both hEGF and EI were maintained at −20°C.
9
Preparation of Drug Stock Solutions
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Trametinib (GSK1120212) and dabrafenib (GSK2118436) were kindly provided by GlaxoSmithKline (Brentford, Middlesex, UK). Gedatolisib (PF05212384) was kindly provided by Pfizer Inc. (New York, NY, USA). SCH772984 and alpelisib were purchased by Selleckchem (Houston, TX, USA). Everolimus was obtained from Novartis Pharma (Basel, Switzerland). MK-2206 was kindly provided by Merck and Co. (Kenilworth, NJ, USA).
Trametinib, dabrafenib, SCH772984, alpelisib and MK-2206 were dissolved in DMSO as 1 mM, 10 mM, 0.2 mM, 10 mM and 1 mM stock solution, respectively and stored at −20 °C. Gedatolisib was dissolved in DMSO as 1 mM stock solution and stored at −80 °C. Everolimus was dissolved in 100% ethanol as a 10 mM stock solution and stored at −20 °C.
The final concentration of drugs was obtained by dilution with culture medium.
Trametinib, dabrafenib, SCH772984, alpelisib and MK-2206 were dissolved in DMSO as 1 mM, 10 mM, 0.2 mM, 10 mM and 1 mM stock solution, respectively and stored at −20 °C. Gedatolisib was dissolved in DMSO as 1 mM stock solution and stored at −80 °C. Everolimus was dissolved in 100% ethanol as a 10 mM stock solution and stored at −20 °C.
The final concentration of drugs was obtained by dilution with culture medium.
10
Immunoblot and Immunofluorescence Analysis
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