HaCaT keratinocytes were cultured in DMEM (PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (PAN Biotech) and 1% antibiotics/antimycotics (PAN Biotech). The generation of HaCaT-EV and HaCaT-shAHR keratinocytes has been previously described [16 (link)]. The culture medium of HaCaT-EV and HaCaT-shAHR keratinocytes was supplemented with G418 (Carl Roth, Karlsruhe, Germany). Normal human epidermal keratinocytes were obtained from PromoCell (Heidelberg, Germany) and cultured in Keratinocyte Growth Medium 2 (PromoCell). All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. UV exposure of cells was carried out by using the BS-02 irradiation chamber (Opsytec Dr. Gröbel, Ettlingen Germany) equipped with individually controllable UVA Actinic and UVB bulbs and respective sensors. For both, UV and sham irradiation, cell culture medium was replaced by PBS. For cell treatment, 6-formylindolo[3,2-b]carbazole (Biomol, Hamburg, Germany), tapinarof, dabrafenib, encorafenib (MedChem Express, Monmouth Junction, NJ, USA), 3’-methoxy-4′-nitroflavone (a kind gift from I. Meyer, Symrise AG, Holzminden, Germany), 7-hydroxyflavone (Sigma-Aldrich, Munich, Germany) and vemurafenib (Selleck Chemicals, Houston, TX, USA) were dissolved in dimethyl sulfoxide.
Encorafenib
Manufactured by MedChemExpress
Sourced in United States
Encorafenib is a small molecule drug candidate that inhibits the BRAF kinase enzyme. It is used for research purposes in the laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Sb590885, by Selleck Chemicals (2 mentions)
Prolene, by Johnson & Johnson (2 mentions)
C57bl 6j wild type male mice, by Charles River Laboratories (2 mentions)
Alzet osmotic pumps, by Charles River Laboratories (2 mentions)
Sotorasib, by MedChemExpress (2 mentions)
Dabrafenib, by MedChemExpress (1 mentions)
Normal human epidermal keratinocytes, by PromoCell (1 mentions)
Keratinocyte growth medium 2, by PromoCell (1 mentions)
7 hydroxyflavone, by Merck Group (1 mentions)
3 methoxy 4 nitroflavone, by Merck Group (1 mentions)
Antibiotics antimycotics, by PAN Biotech (1 mentions)
Tapinarof, by MedChemExpress (1 mentions)
Vemurafenib, by Selleck Chemicals (1 mentions)
Dulbecco modified eagle medium (dmem), by PAN Biotech (1 mentions)
Fetal bovine serum (fbs), by PAN Biotech (1 mentions)
Ouabain, by MedChemExpress (1 mentions)
Paraformaldehyde fix solution, by Beyotime (1 mentions)
Annexin 5 pi double staining, by BD (1 mentions)
Transwell cell culture inserts, by Merck Group (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
6 protocols using encorafenib
1
Culturing and Treating HaCaT Keratinocytes
2
Investigating Cytotoxic Effects of Ouabain and Encorafenib
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The chemicals ouabain and encorafenib were purchased from MedChem Express (Princeton, NJ, USA). Cell counting kit-8 (CCK8), crystal violet staining solution, 4% paraformaldehyde fix solution, and cell cycle and apoptosis analysis kit were purchased from Beyotime Biotechnology (Shanghai, China). The apoptosis detection kit (Annexin V-PI double staining) was purchased from BD Biosciences (San Jose, CA, USA). Transwell cell culture inserts with 8 μM diameter were purchased from Millipore Corporation (Bedford, MA, USA). The antibodies against Bcl-2, Bax, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA).
3
Osmotic Pump Delivery of Inhibitors in Mice
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Male wild-type C57Bl/6J mice (7–8 weeks of age) were from Charles River (U.K.). Drug delivery used Alzet osmotic pumps (models 1007D or 1004; supplied by Charles River), filled according to the manufacturer's instructions in a laminar flow hood using sterile technique. Mice were treated with vehicle only [DMSO/PEG mix: 50% (v/v) DMSO, 20% (v/v) polyethylene glycol 400, 5% (v/v) propylene glycol, 0.5% (v/v) Tween 80 made up to 100% with H2O], or 0.5 mg/kg/d SB590885 (Selleck Chemicals) or 3 mg/kg/d encorafenib (MedChemExpress) dissolved in DMSO/PEG mix. In some studies, mice also received a minipump containing angiotensin II (AngII) for delivery at 0.8 mg/kg/d or acidified PBS vehicle. Minipumps were incubated overnight in sterile PBS (37°C) prior to implantation. Implantation was performed under continuous inhalation anaesthesia using isoflurane (induction at 5%, maintenance at 2–2.5%) mixed with 2 l/min O2. A 1 cm incision was made in the mid-scapular region and mice were given 0.05 mg/kg (s.c.) buprenorphine (Ceva Animal Health Ltd.) to repress post-surgical discomfort. Minipumps were implanted portal first in a pocket created in the left flank region of the mouse. Wound closure used a simple interrupted suture with polypropylene 4-0 thread (Prolene, Ethicon). Mice were allowed to recover singly and returned to their home cage once fully recovered.
4
Cell Line Maintenance and Compound Acquisition
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The pancreatic cancer cell line MIA PaCa-2 was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). The lung cancer cell lines LU65 and NCI-H23 were obtained from the RIKEN Cell Bank (Ibaraki, Japan) and American Type Culture Collection (Manassas, VA, USA), respectively. MIA PaCa-2 cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (FBS), 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate. NCI-H23 and LU65 cells were maintained in Roswell Park Memorial Institute 1640 medium supplemented with 10% FBS. In all culture media, 100 U/mL penicillin and 100 μg/mL streptomycin were added to prevent bacterial contamination. All cell culture reagents were purchased from Gibco, Thermo Fisher Scientific (Waltham, MA, USA). The cell lines were incubated in a humidified incubator at 37 °C under 5% CO2 and 95% air atmosphere. All cells were verified through short-tandem repeat (STR) profiling and tested negative for mycoplasma contamination.
Sotorasib, ARS-1620, Q-VD-OPh (QVD), cobimetinib, N-acetyl-l-cysteine (NAC), and encorafenib were purchased from MedChemExpress (Monmouth Junction, NJ, USA).
Sotorasib, ARS-1620, Q-VD-OPh (QVD), cobimetinib, N-acetyl-l-cysteine (NAC), and encorafenib were purchased from MedChemExpress (Monmouth Junction, NJ, USA).
5
Implantation of Alzet Osmotic Pumps in Mice
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Male wild-type C57Bl/6J mice (7-8 weeks of age) were from Charles River (UK).
Drug delivery used Alzet osmotic pumps (models 1007D or 1004; supplied by Charles River), filled according to the manufacturer's instructions in a laminar flow hood using sterile technique. Mice were treated with vehicle only [DMSO/PEG mix: 50% (v/v) DMSO, 20% (v/v) polyethylene glycol 400, 5% (v/v) propylene glycol, 0.5% (v/v) Tween 80 made up to 100% with H 2 O], or 0.5 mg/kg/d SB590885 (Selleck Chemicals) or 3 mg/kg/d encorafenib (MedChemExpress) dissolved in DMSO/PEG mix. Minipumps were incubated overnight in sterile PBS (37°C) prior to implantation. Implantation was performed under continuous inhalation anaesthesia using isoflurane (induction at 5%, maintenance at 2 -2.5%) mixed with 2 l/min O 2 . A 1 cm incision was made in the mid-scapular region and mice were given 0.05 mg/kg (s.c.) buprenorphine (Ceva Animal Health Ltd.) to repress post-surgical discomfort. Minipumps were implanted portal first in a pocket created in the left flank region of the mouse. Wound closure used a simple interrupted suture with polypropylene 4-0 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. thread (Prolene, Ethicon). Mice were allowed to recover singly and returned to their home cage once fully recovered.
Drug delivery used Alzet osmotic pumps (models 1007D or 1004; supplied by Charles River), filled according to the manufacturer's instructions in a laminar flow hood using sterile technique. Mice were treated with vehicle only [DMSO/PEG mix: 50% (v/v) DMSO, 20% (v/v) polyethylene glycol 400, 5% (v/v) propylene glycol, 0.5% (v/v) Tween 80 made up to 100% with H 2 O], or 0.5 mg/kg/d SB590885 (Selleck Chemicals) or 3 mg/kg/d encorafenib (MedChemExpress) dissolved in DMSO/PEG mix. Minipumps were incubated overnight in sterile PBS (37°C) prior to implantation. Implantation was performed under continuous inhalation anaesthesia using isoflurane (induction at 5%, maintenance at 2 -2.5%) mixed with 2 l/min O 2 . A 1 cm incision was made in the mid-scapular region and mice were given 0.05 mg/kg (s.c.) buprenorphine (Ceva Animal Health Ltd.) to repress post-surgical discomfort. Minipumps were implanted portal first in a pocket created in the left flank region of the mouse. Wound closure used a simple interrupted suture with polypropylene 4-0 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. thread (Prolene, Ethicon). Mice were allowed to recover singly and returned to their home cage once fully recovered.
6
Synthesis and Evaluation of BRAF Inhibitors
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IHMT-RAF-128 was in-house synthesized following the route shown in Scheme 1; Vemurafenib (PLX4032), LY3009120, Encorafenib, Adagrasib, Sotorasib, and RAF709 were purchased from MedChemExpress (Shanghai, P.R.China).
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