The following antibodies were used: anti-FLAG (Sigma, F7425), anti-IKZF3 (Cell Signaling, #15103), anti-IKZF1 (Cell Signaling, #9034), anti-RUNX1 (Abcam, ab92336), anti-RUNX3 (Cell Signaling, #9647), anti-CBFβ (Santa Cruz, sc-20693), anti-CRBN (Sigma, HPA045910), anti-ubiquitin (K48) (EMD Millipore, #05–1307), anti-TUBULIN (Santa Cruz, sc-8035), anti-VINCULIN (Santa Cruz, sc-73614), anti-Rabbit IgG-HRP (GE Healthcare, NA934V), and anti-mouse IgG-HRP (GE Healthcare, NA931V). The following agarose beads were used: anti-FLAG M2 Affinity Gel (Sigma, A2220) and Glutathione Sepharose 4B (GE Healthcare, #17075601). The following in vitro translation kit was used: TNT T7 Coupled Reticulocyte Lysate Systems (Promega, L4610). Benzonase was used according to manufacturer (Sigma, E1014). The following compounds were used: Lenalidomide (Sigma, CDS022536), Pomalidomide (Sigma, P0018), Bortezomib (Millennium Pharmaceuticals), and AI-10–104 was kindly provided by Dr. John H. Bushweller.
Lenalidomide
Manufactured by Merck Group
Sourced in United States
Lenalidomide is a pharmaceutical compound used in laboratory research and development. It functions as an immunomodulatory agent, capable of modulating the immune system. Lenalidomide is commonly utilized in cell culture experiments and preclinical studies across various therapeutic areas.
Automatically generated - may contain errors
Lab products found in correlation
Pomalidomide, by Merck Group (6 mentions)
Mg132, by Merck Group (4 mentions)
Cycloheximide (chx), by Merck Group (4 mentions)
Bortezomib, by Selleck Chemicals (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Bortezomib, by Merck Group (3 mentions)
Anti ikzf3, by Cell Signaling Technology (2 mentions)
Anti ikzf1, by Cell Signaling Technology (2 mentions)
Anti runx1, by Abcam (2 mentions)
Anti cbfβ, by Santa Cruz Biotechnology (2 mentions)
Anti crbn, by Merck Group (2 mentions)
Anti ubiquitin k48, by Merck Group (2 mentions)
Anti vinculin, by Santa Cruz Biotechnology (2 mentions)
Anti rabbit igg hrp, by GE Healthcare (2 mentions)
Anti mouse igg hrp, by GE Healthcare (2 mentions)
Tnt t7 coupled reticulocyte lysate system, by Promega (2 mentions)
Anti tubulin, by Santa Cruz Biotechnology (2 mentions)
Anti runx3, by Cell Signaling Technology (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti flag m2 affinity gel, by Merck Group (2 mentions)
26 protocols using lenalidomide
1
Characterizing CRBN-mediated Protein Degradation
2
Myeloma Cytotoxicity Dose-Response Assays
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For dose–response cell toxicity assays, 1 E+3 myeloma cells were seeded per well in 384-well plates (Corning) using the Multidrop Combi (Thermo Fisher) and incubated for 24 h. In monotherapy cytotoxicity assays, cells were treated with drug or DMSO and incubated for 48 h, while cells were further incubated with E7107 (H3) for an additional 24 h in E7107 dual therapy combination assays. Cfz (Selleckchem, S2853), melphalan (Sigma, S2853), Bortezomib (Selleckchem, S1013), Venetoclax (LC Labs, V-3579), Lenalidomide (Sigma, CDS022536), E7107 (H3 Biomedicine, CAS:630100–90–2), and KH-CB19 (sc-362756) were solubilized in DMSO at 10 mM.
All cell viability was determined with Cell-Titer Glo reagent (Promega, G7573) using a Glomax Explorer (Promega) luminescence plate reader. For the drug titration cytotoxicity assays, measurements were performed in quadruplicate, while measurements were performed in triplicate in all other assays, and viabilities are reported as mean (+/−S.D.) ratio normalized to DMSO-treated controls or measurements at 0 h. For ZIP synergy calculations, normalized viability data were submitted to SynergyFinder web application44 (link).
All cell viability was determined with Cell-Titer Glo reagent (Promega, G7573) using a Glomax Explorer (Promega) luminescence plate reader. For the drug titration cytotoxicity assays, measurements were performed in quadruplicate, while measurements were performed in triplicate in all other assays, and viabilities are reported as mean (+/−S.D.) ratio normalized to DMSO-treated controls or measurements at 0 h. For ZIP synergy calculations, normalized viability data were submitted to SynergyFinder web application44 (link).
3
Murine Myeloma Cell Line Maintenance and PD-L1 Expression Assay
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NS-1 and MOPC-315 murine myeloma cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The NS-1 cells were maintained in RPMI 1640 medium, and the MOPC-315 cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% of fetal bovine serum and 1% anti-anti solution. Both cell lines were sub-cultured every 2-3 days. Lenalidomide (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO, and the stock solution was stored at –70°C. Phycoerythrin- (PE) conjugated anti-mouse PD-L1 antibody was purchased from BD Bioscience (San Jose, CA, USA) and used as a control antibody for the mouse PD-L1 expression assays.
4
Modulating PBMC Activation Pathways
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The 3 × 106 PBMCs from healthy donors or individuals carrying heterozygous IKZF1 mutation were cultured in the presence poly(I:C) (10 μg/ml, Invivogen), LPS (5ng/ml, Sigma), CL075 (1 μg/ml, Invivogen) and CpG (ODN 2216, 7.5 μΜ, Invivogen) with or without IFN-α (3000 IU/ml, R&D), with or without anti-CD303 and anti-CD304 (Biolegend), with or without 0.1, 1 or 10 μM lenalidomide (Sigma). Cells were cultured for 14 h at 37 oC, 5% CO2, with addition of Brefaldin A (10 μg/ml, eBioscience) after 3 h. For dead-cell exclusion (usually <30%) cells were stained with Zombie amine dye (Biolegend), surface markers and then intracellular cytokines antibodies after fixation and permeabilization (eBioscience), as above.
5
Evaluation of Proteasome Inhibitors in Myeloma
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Antibodies against IκBα (C-21), IκBβ (C-20) were obtained from Santa Cruz Biotechnology and antibody against tubulin (CP06) purchased from Calbiochem, recombinant human TNFα (654205, EMD Millipore), cycloheximide (C7698, Sigma-Aldrich), and leptomycin B (87081-35-4, Cayman Chemicals). Bortezomib (S1013), ixazomib citrate (S2181), carfilzomib (S2853), melphalan (S8266), and bendamustine (S1212) were purchased from Selleckchem. Dexamethasone (D4902), doxorubicin (D1515), and lenalidomide (CDS022536) were purchased from Sigma-Aldrich. Selinexor (KPT-330) was provided by Dr. Trinayan Kashyap (Karyopharm). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (M6494) was purchased from Thermo-Fisher Scientific.
6
Natural Product Library Screening Protocol
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The natural product library was obtained from Target Molecule Corp., Wellesley Hills, MA. Nam was provided by MedChemExpress LLC, Princetion, NJ. Doxorubicin, lenalidomide, and MG132 were purchased from Sigma-Aldrich Chemicals Co. The antibodies included: anti-HA (M180-3), anti-Myc (M192-3), and anti-Flag (M185-3L) that were from MBL Biotech Co., Ltd. (Beijing, China). Anti-Otub1 (Ab175200) was from Abcam Shanghai, China. Anti-GAPDH (60004-1-Ig), anti-ITGB7 (18309-1-AP), and anti-c-Maf (55013-1-AP) were from Proteintech Group, Inc. (Wuhan, China). Anti-c-Maf (sc-7866) and anti-Ub (sc-271289) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-PARP (#9532), anti-CCND2 (#3741), and anti-Caspase-3 (#9662) were from Cell Signaling Technology, Danvers, MA. Anti-β-tubulin (AC008) was obtained from ABclonal Biotechnology Co., Ltd, Woburn, MA.
7
Cell Surface Proteomics of Myeloma Cells
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For cell surface proteomics experiments, 25e6 cells were seeded at 1e6/mL in T75 flasks and treated with compounds for 48 h at the LD25 dose. RPMI-8226 cells were treated with 50 μM Lenalidomide (Sigma CDS022536), 7.5 nM Bortezomib (Selleck Chemicals, S1013), or 300 nM CB-5083 (gift of Cleave Biosciences). AMO1 cells were treated with 50 μM Lenalidomide, 750 nM JG-342 (gift of Jason Gestwicki, UCSF), or 250 nM CB-5083. For flow cytometry experiments, cells were plated and treated in 96-well plates with the doses described in figure legends for 48 h prior to flow cytometry analysis unless otherwise indicated. For Bortezomib drug screens, 1000 cells were seeded in 45 μL of media in 384-well plates using the Multidrop Combi (Thermo Scientific). Bortezomib (Selleck Chemicals, S1013) was added 24 h after seeding and viability was measured using Cell-Titer Glo (Promega) 48 h after the addition of drugs using a GlowMax Explorer (Promega).
8
Expansion of CD34+ Bone Marrow Progenitors
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CD34+ bone marrow progenitors were purified by fluorescence-activated cell sorting (FACS) (>98% purity) and seeded (3000/well) onto OP9 stromal cells (5000/well) in 96-well U-bottomed plates. Cells were cultured in 200 μl αMEM (Gibco™) supplemented with 1% penicillin/streptomycin (Sigma), 10% fetal calf serum (Gibco), 20 ng/ml granulocyte-macrophage colony-stimulating factor (R&D systems), 100 ng/ml Flt3-ligand (Immunotools), 20 ng/ml stem cell factor (Immunotools), with or without 0.1, 1 or 10 μM lenalidomide (Sigma) or 0.01% dimethyl sulfoxide control. Half the volume of media, with cytokines, was replaced weekly. At day 22, cells were harvested on ice, passed through a 50 μm filter, washed and stained for flow cytometric analysis.
9
IMiDs Treatment Optimization Protocol
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Lenalidomide and pomalidomide are from Sigma-Aldrich. IMiDs were dissolved at the concentration of 80 mM in DMSO (Sigma-Aldrich) and stored at −80°C until further use. Except for dose-effect experiments, IMiDs were used at a final concentration of 10 µM. In all experiments, corresponding concentrations of DMSO were used as a negative control. Recombinant human (rh) IL-2 was purchased from Novartis, and rh-IL-15 from R&D systems.
10
Labeled Mcl-1 Protein Production
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Oxcarbazepine, Risperidone, Lenalidomide and Torsemide were purchased from Sigma-Aldrich and Deferasirox from VWR. Soluble labelled 15N Mcl-1 (172–327) was produced following a previously published protocol.36 (link)
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