Obinutuzumab (GA101) was generously supplied by Hoffmann La Roche (Switzerland). RTX was purchased from Genentech Inc. (South San Francisco, CA, USA). Antibodies for flow cytometry, CD20-PE (Clone HI47), CD20-FITC (Clone HI47) and CD19-FITC (SJ25-C1), were obtained from Invitrogen (CA, USA). Western blot antibodies, phospho-PLC gamma2 (Tyr759, 150 KDa), total PLC gamma2 (150 KDa), phospho GSK3 Beta (Ser21/9, 46 KDa), total GSK3 Beta (46KDa), phospho BTK (Tyr223, 78KDa), total BTK (Clone D3H5, 77KDa), phospho Lyn (Tr507, 53, 56KDa) and total Lyn (C13F9,58KDa), phospho B- RAF (Ser445, 86KDa), total B- RAF (clone 55C6, 86KDa), phospho ERK1/2 (Thr202/Thyr204) (Clone D1.14.4E, 42, 44KDa), total ERK1/2 (42,44KDa), phospho LCK (Tyr505, 56KDa), total LCK (56KDa) and B-actin (clone D6A8, 45KDa) were obtained from Cell Signaling Technology (Danvers, MA, USA). CD20 (B9E9) antibody was obtained by Santa Cruz Biotechnology (Dallas, TX, USA). Annexin V: PE Apoptosis Detection Kit I was obtained from BD Biosciences (San Jose, CA, USA). Alamar blue cell viability reagent was purchased from Invitrogen (Grand Island, NY, USA). U0126 MEK1/2 inhibitor was purchased from Cell Signaling Technology (Danvers, MA, USA). PLC gamma2 and GSK3 Beta ON-TARGET plus siRNA were obtained from Dharmacon GE (Lafayette, CO, USA).
Mek1 2 inhibitor u0126
Manufactured by Cell Signaling Technology
Sourced in United States
MEK1/2 inhibitor U0126 is a highly selective and potent inhibitor of the mitogen-activated protein kinase (MAPK) kinases MEK1 and MEK2. U0126 inhibits the activation of ERK1 and ERK2 by blocking the phosphorylation of these kinases by upstream MEK1 and MEK2. This compound is commonly used in cell-based assays and research applications to study the role of the MAPK/ERK signaling pathway.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Phospho lyn, by Cell Signaling Technology (1 mentions)
Phospho gsk 3 beta, by Cell Signaling Technology (1 mentions)
Phospho btk, by Cell Signaling Technology (1 mentions)
Total btk, by Cell Signaling Technology (1 mentions)
Phospho b raf, by Cell Signaling Technology (1 mentions)
Total lck, by Cell Signaling Technology (1 mentions)
Total erk1 2, by Cell Signaling Technology (1 mentions)
Alamarblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Pe annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Pyrrophenone, by Cayman Chemical (1 mentions)
Fetal calf serum (fcs), by GE Healthcare (1 mentions)
Tnfα neutralizing antibody, by Cell Signaling Technology (1 mentions)
Dulbecco s pbs, by Thermo Fisher Scientific (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Minimum essential medium (mem), by Merck Group (1 mentions)
11 protocols using mek1 2 inhibitor u0126
1
Obinutuzumab and Rituximab Signaling Analysis
2
Cytokine Assay Protocol for Immune Cells
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Glutamine, penicillin-streptomycin-nystatin, phosphate buffered saline (PBS) Dulbecco’s Modified Eagle’s Medium (DMEM), Hanks’ Balanced Salts Solution (HBSS), fetal bovine serum (FBS), HEPES, sodium pyruvate, Dulbecco’s Modified Eagle’s/F12 (HAM) medium (DMEM/F12) were from Beth Ha-Emek, Biological Industries, Israel.
Sodium azide, trypan blue, p-nitrophenylphosphate, phenylmethylsulfonyl fluoride, leupeptin, benzamidine, aprotinin, DMSO, Tween 20, Tris, 4,6-diamidino-2-phenylindole (DAPI), bovine serum albumin (BSA), Trypsin-EDTA, dihyroethidium (DHE), lipopolysaccharide (LPS), Skim Milk Powder, Poly-L-lysine, horseradish peroxidase (HRP), 1,2-Dioleoyl-sn-glycerol, Triton X-100, β-mercaptoethanol, Percoll, non-essential amino-acids, Diphenyliodonium chloride (DPI) were from Sigma Israel, Rehovot, Israel. Fetal calf serum was from GE Healthcare Life Sciences HyClone Laboratories, Inc., Logan Utah, USA. ECL detection kit for the immunoblot analysis was from PerkinElmer, MA, USA. Pyrrophenone was from Cayman Chemical, Michigan, USA. TNF-α-neutralizing antibody and U0126 (MEK1/2 inhibitor) were from Cell Signaling Technology, Danvers, MA, USA. Interleukin (IL)-4, IL-10, TNF-α, IFN-γ were from PeproTech Asia, NJ, USA.
Sodium azide, trypan blue, p-nitrophenylphosphate, phenylmethylsulfonyl fluoride, leupeptin, benzamidine, aprotinin, DMSO, Tween 20, Tris, 4,6-diamidino-2-phenylindole (DAPI), bovine serum albumin (BSA), Trypsin-EDTA, dihyroethidium (DHE), lipopolysaccharide (LPS), Skim Milk Powder, Poly-L-lysine, horseradish peroxidase (HRP), 1,2-Dioleoyl-sn-glycerol, Triton X-100, β-mercaptoethanol, Percoll, non-essential amino-acids, Diphenyliodonium chloride (DPI) were from Sigma Israel, Rehovot, Israel. Fetal calf serum was from GE Healthcare Life Sciences HyClone Laboratories, Inc., Logan Utah, USA. ECL detection kit for the immunoblot analysis was from PerkinElmer, MA, USA. Pyrrophenone was from Cayman Chemical, Michigan, USA. TNF-α-neutralizing antibody and U0126 (MEK1/2 inhibitor) were from Cell Signaling Technology, Danvers, MA, USA. Interleukin (IL)-4, IL-10, TNF-α, IFN-γ were from PeproTech Asia, NJ, USA.
3
Characterization of Signaling Pathways
4
Investigating Cell Line Responses
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Human colonic myofibroblast cells CCD18COCo (ATCC CRL1459), colon adenocarcinoma cells HT-29 (ATCC HTB-38), lung cancer cells A549 (ATCC CCL185) and breast cancer cells MCF7 (ATCC HTB22) were obtained from ATCC (LGC Standards, Middlesex, UK) and bladder cancer cells EJ138 were obtained from ECACC (Public Health England, Salisbury, UK). All cells were used within 6 months of resuscitation and were authenticated by the ATCC or ECACC by using standard protocols prior to purchase. Cells were cultured in minimum essential medium from Sigma-Aldrich (Dorset, UK) supplemented with 10 % fetal bovine serum (Labtech International, East Sussex, UK). Human colonic myofibroblast cells were used up to passage 10. The human recombinant HMGB1, anti-HMGB1 primary antibody, anti-RAGE primary antibody and anti-TLR4 primary antibody were purchased from R&D Systems (Oxford, UK). MEK1/2 inhibitor (U0126) and PI3K inhibitor (LY294002) were purchased from Cell Signaling Technology (Massachusetts, USA). Protease and phosphatase cocktail set inhibitors were purchased from Calbiochem (Nottingham, UK). All other reagents were obtained from Fisher Scientific (Leicestershire, UK), BioRad Laboratories (Hertfordshire, UK) or Sigma-Aldrich.
5
Signaling Pathway Modulation Protocol
6
Comprehensive Antibody Acquisition for Protein Analysis
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Anti-p53, anti-phospho-Stat-3 and total Stat-3 antibodies were acquired from Santa Cruz Biotechnology; anti-MMP-14 antibodies were acquired from Millipore; anti-Sp1 and anti-MDM2 were acquired from Abcam; anti-myc tag was acquired from Roche; anti-phospho-ERK 1/2 and anti-total ERK antibodies were acquired from Sigma-Aldrich; anti-phospho-AKT and anti-total AKT antibodies were obtained from Cell Signaling. Anti-actin antibodies were attained from Cell Signaling Technologies and anti-IgG was obtained from Millipore for probing loading or experimental setup controls, respectively. Horseradish-peroxidase (HRP) conjugated anti-mouse and anti-rabbit secondary antibodies were acquired from Rockland Immunochemicals. Hoechst nuclear stain was acquired from Invitrogen. Proteasome inhibitor MG-132 and protein synthesis inhibitor cycloheximide were acquired from Sigma-Aldrich. Recombinant IL-6 was purchased from either Sigma-Aldrich or Gold Biotechnology. STAT3 Inhibitor XVIII, BP-1-102 and PI3K inhibitor LY294002 were acquired from Calbiochem; MEK1/2 inhibitor U0126 was acquired from Cell Signaling Technologies.
7
Signaling Pathway Inhibition in Melanoma Cells
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Melanoma cell lines WM239 and WM164 were cultured in RPMI 1640 medium (Gibco‐BRL, UK). SK‐Mel‐28 and Bowes were cultured in minimum essential medium (MEM) and Eagle's minimum essential medium (EMEM) (Invitrogen, Carlsbad, CA). In experiments where signalling pathways were inhibited, the cells were cultured for 3 days in a medium containing 10 μm PI3 kinase inhibitor LY294002 (Tocris Bioscience, Bristol, UK), MEK 1/2 inhibitor U0126 (Cell Signaling Technology, Danvers, MA) or BRAF inhibitor vemurafenib (Santa Cruz Biotechnology, Santa Cruz, CA). PI3K pathway inhibition was verified by immunoblotting with p‐Akt and Akt antibodies (Cell Signaling Technology) and MAPK pathway inhibition by immunoblotting with a p‐ERK 1/2 antibody (Cell Signaling Technology) and an ERK 2 antibody (Santa Cruz Biotechnology), all produced in rabbit.
8
Evaluating Cell Viability with Inhibitors
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SH-SY5Y cells seeded in 96-well plates were cultured overnight to form a confluent monolayer. Cells were cultured for 24 h in fresh culture medium containing various concentrations of MEK1/2 inhibitor U0126 (Cell Signaling Technology) or AKT inhibitor MK-2206 (Selleck) dissolved in dimethylsulfoxide (DMSO; Sigma). Cell viability was evaluated using CellTiter 96®AQueous One Solution Cell Proliferation kit (Promega) following the manufacture's instructions. The absorbance at 490 nm was read on a Synergy 2 Multi-Mode Microplate Reader (BioTek).
9
CCL21-Induced VEGF-C Secretion
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Control and CCR7 shRNA MDA-MB-231 cells were cultured in serum-free media and treated with or without human CCL21/6Ckine (350 ng/ml, R&D Systems, Minneapolis, MN, USA) for 24 hours before supernatants were collected. Then, VEGF-C concentration in conditioned media was measured by ELISA (Quantikine Human VEGF-C Immunoassay, R&D Systems). For signaling studies, cells were pretreated with various concentrations of PI3 kinase inhibitor (LY294002) (Cell Signalling Technology, Danvers, MA, USA), AKT inhibitor (Akti-1/2) (Abcam, Cambridge, MA, USA), and MEK 1/2 inhibitor (U0126) (Cell Signalling). Tumor cells were then treated with CCL21/6Ckine for 24 hours and the level of VEGF-C secretion was determined. In order to quantify CCL21 protein secretion, MDA-MB-231 and HMVEC-dLy cells were maintained in 2D and 2D-matrix culture conditions and subjected to Quantikine Human 6Ckine Immunoassay (R&D Systems).
10
HER2-amplified breast cancer cell lines and signaling inhibition
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Human HER2-amplified breast cancer cell lines MDA-MB-453 and SK-BR-3 were obtained from the American Type Culture Collection (USA). The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Lonza, Switzerland) supplemented with 10% fetal bovine serum (FBS; Lonza), 5 mM ultraglutamine, and 100 U/mL penicillin-streptomycin (Gibco, USA). The cells were grown in an incubator at 37°C with 5% CO2 and periodically checked for mycoplasma contamination using a MycoAlertTM mycoplasma detection kit (Lonza).
In experiments where signaling pathways were inhibited, the cells were kept in starvation medium (DMEM 0.5% FBS) overnight and then cultured for 24 hours in a medium containing 10 μM MEK 1/2 inhibitor U0126 (Cell Signaling Technology, USA), 10 μM PI3 kinase inhibitor LY294002 (Tocris Bioscience, UK), or DMSO (Thermo Fisher Scientific) as a negative control.
In experiments where signaling pathways were inhibited, the cells were kept in starvation medium (DMEM 0.5% FBS) overnight and then cultured for 24 hours in a medium containing 10 μM MEK 1/2 inhibitor U0126 (Cell Signaling Technology, USA), 10 μM PI3 kinase inhibitor LY294002 (Tocris Bioscience, UK), or DMSO (Thermo Fisher Scientific) as a negative control.
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