Melanoma cells were seeded into 96-well plates in a density of 1.5 × 103 cells per well. After 24 h cells were treated with Raf inhibitors Encorafenib (Selleckchem LGX818, #S7108) or Vemurafenib (Selleckchem PLX4032, RG7204, #S1267) and MEK inhibitor Binimetinib (Selleckchem MEK162, ARRY-162, ARRY-438162, #S7007) in different concentrations and incubated for 72 h. Proliferation and viability of the cells was determined with a standard colorimetric assay using 7-hydroxy-3H-phenoxazin-3-one-10-oxide sodium salt (Sigma Resazurin sodium salt, #R7017), where the bioreduction of the dye was measured at 595 nm. IC50 values were determined by nonlinear regression using the dose–response equations built into GraphPad Prism software version 5 (GraphPad Software Inc., USA).
Encorafenib
Manufactured by Selleck Chemicals
Sourced in United States
Encorafenib is a laboratory reagent used in research and development. It is a small-molecule inhibitor that targets the BRAF kinase. Encorafenib is utilized in various scientific investigations and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trametinib, by Selleck Chemicals (8 mentions)
Vemurafenib, by Selleck Chemicals (7 mentions)
Dabrafenib, by Selleck Chemicals (6 mentions)
Tak 632, by Selleck Chemicals (6 mentions)
Binimetinib, by Selleck Chemicals (5 mentions)
Cobimetinib, by Selleck Chemicals (4 mentions)
Ly3009120, by Selleck Chemicals (3 mentions)
Efavirenz, by Selleck Chemicals (2 mentions)
Mek162, by Selleck Chemicals (2 mentions)
Zidovudine, by Selleck Chemicals (2 mentions)
Stavudine, by Selleck Chemicals (2 mentions)
Resazurin sodium salt, by Merck Group (1 mentions)
Prism software, by GraphPad (1 mentions)
Q vd oph, by Merck Group (1 mentions)
Raf265, by Selleck Chemicals (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Vemurafenib, by Roche (1 mentions)
Saracatinib, by Selleck Chemicals (1 mentions)
Ulixertinib, by Selleck Chemicals (1 mentions)
Imatinib, by Cayman Chemical (1 mentions)
14 protocols using encorafenib
1
Evaluating Raf and MEK Inhibitors on Melanoma Cells
2
Synergistic BRAF and MEK Inhibitor Evaluation
3
Inhibitor Concentrations Across Studies
4
Inhibitor Preparation for Cell Studies
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TAK-632 (#S7291), Vemurafenib (PLX4032, #1267), and Encorafenib (LGX818, #S7108) were purchased from Selleck Chemicals LLC, Houston, TX, USA. Sorafenib tosylate (Axon 1397) was from Axon Medchem, Groningen, The Netherlands, and SB-590885 (2650/10) was purchased from R&D Systems, Minneapolis, MN, USA. Inhibitors were dissolved in DMSO. Stocks were kept at −80 °C and further diluted in DMSO as required.
5
Inhibitor Preparation and Storage
6
Kinase Inhibitor Library Characterization
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Kinase inhibitors library was obtained from Selleck. Vemurafenib, Encorafenib, Trametinib, TAK-632, AZ-628, LY3009120, Lapatinib, and Ponatinib were purchased from Selleck and purity >99% was confirmed by NMR and MS. PHI1 and PHI2 compounds were synthesized in >99% purity and confirmed by NMR and MS. All compounds were dissolved in DMSO to a 10 mM stock solution.
7
Evaluating Metastatic Melanoma Cell Lines
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Melanoma cell lines Malme3M (lung metastasis, BRAFmut), WM3734 (brain metastasis, BRAFmut), and WM1366 (primary melanoma, vertical growth phase, NRASmut) were purchased from ATCC (Manassas, VI, USA) and Rockland Immunochemicals, Inc. (Pottstown, PA, USA) and were subject to regular tests for excluding Mycoplasma contamination. Malme3M was cultured in RPMI + 20% fetal bovine serum (FBS), WM3734 and WM1366 were cultured in RPMI + 10% FBS. Encorafenib, binimetinib, vemurafenib, cobimetinib, dabrafenib, and trametinib were purchased from Selleckchem and dissolved in dimethylsulfoxide (DMSO).
8
Melanoma Cell Line Combination Assays
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Human melanoma cell lines M14, A375 and WM115 were obtained as previously described [9 (link), 12 (link)] and cultured in RPMI supplemented with 10% FBS. For combination assays, BRAFi has been used starting from the highest dose of 5 μM and then diluted 1:2 for 10 times; SPV122 was used at fixed dose of 1.25 μM. All the other experiments have been performed with the following doses: 150 nM for BRAFi, 75 nM for MEKi and 1.25 μM for SPV122. Long-term colony formation assays have been performed treating M14 melanoma cells two times a week with 250 nM of a BRAFi and at every time point cells have been fixed using crystal violet staining as previously done [10 (link)]. For clonogenic assays, Zidovudine and Stavudine NRTIs and SPV122 and Efavirenz NNRTIs have been tested starting from 100 μM and then diluted 1:2 for 10 times. Encorafenib as BRAFi, MEK162 as MEKi, Efavirenz, Stavudine and Zidovudine have been obtained by Selleckchem. SPV122 was prepared as described [29 (link)].
9
Comprehensive BRAF and MAPK Signaling Assays
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A375, SK-Mel28, WM-793, and SK-Mel2 were obtained from ATCC and were validated by sequencing before initiation of the study. All cells were grown in RPMI media (Gibco) supplemented with 10% foetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco). Media for the resistant cells contains 2 μM PLX4032 (Selleck chemicals). HeLa cells were also validated by sequencing and grown in DMEM (Gibco) 10% foetal bovine serum and 2 mM L-glutamine. The cells were maintained at 37°C and 5% CO2. Cells were transfected with Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol and the amount of DNA and siRNA is indicated in each experiment. FLAG-BRAF, -BRAFR509H, -BRAF V600E/R509H, and BRAFV600E, MYC-LATS1, and FLAG-MST2 have been described (Romano et al, 2014a (link); Jambrina et al, 2016 (link); Quinn et al, 2021 (link)) HA-ubiquitin was a gift from Edward Yeh (plasmid # 18712; Addgene) (Kamitani et al, 1997 (link)). MST2 SMARTpool siRNA is from Dharmacon (M-012200) and was validated before (Matallanas et al, 2007 (link)). Bortezomib (S1013), trametinib (S2673), dabrafenib (GSK2118436), encorafenib (LGX818), sorafenib tosylate (S1014), and TAK-632 are from Selleck Chemicals and Proteasome inhibitor I from Calbiochem (539160).
10
Evaluating Targeted Therapies for Cancer Cell Lines
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The melanoma cells A375, lung cancer cells NCI-H1395 (H1395), and colon cancer cells SW620 were purchased from the American Type Culture Collection (ATCC). SKMel246 melanoma cells came from the Memorial Sloan Kettering Cancer Center (MSK) cell collection. JVM3, a BRAF mutant chronic lymphocytic leukemia (CLL) cell line, was obtained from Dr. Omar Abdel-Wahab at MSK. CLR-36 colon cancer cells were generated from a patient-derived xenograft (PDX) by PDX tumor tissue dissociation in vitro and then expansion in culture medium. All cells were grown in media supplemented with penicillin, streptomycin, and 10% fetal bovine serum. Cells were grown in the following media: A375 (DMEM), JVM3 (RPMI), SW620 (RPMI), H1395 (RPMI), SKMel246 (RPMI), and CLR-36 (McCoy’s).
Immunoblotting was performed as previously described.13 (link) Antibodies against ERK, phospho-ERK, phospho-MEK, and GAPDH were purchased from Cell Signaling Technology. Anti-DUSP6 antibody was purchased from abcam.
Encorafenib, binimetinib, and SCH772984 were purchased from SelleckChem. PLX4720 was obtained from Plexxikon. Drugs for in vitro studies were dissolved in dimethyl sulfoxide (DMSO) to yield 10 mM or 1 mM stock solutions and stored at −20°C.
Immunoblotting was performed as previously described.13 (link) Antibodies against ERK, phospho-ERK, phospho-MEK, and GAPDH were purchased from Cell Signaling Technology. Anti-DUSP6 antibody was purchased from abcam.
Encorafenib, binimetinib, and SCH772984 were purchased from SelleckChem. PLX4720 was obtained from Plexxikon. Drugs for in vitro studies were dissolved in dimethyl sulfoxide (DMSO) to yield 10 mM or 1 mM stock solutions and stored at −20°C.
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