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Panobinostat

Manufactured by LC Laboratories
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Panobinostat is a non-selective histone deacetylase (HDAC) inhibitor. It is used for research purposes in the field of epigenetics and oncology.

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Lab products found in correlation

Vorinostat, by Cayman Chemical (4 mentions) Cycloheximide (chx), by Enzo Life Sciences (3 mentions) Bortezomib, by LC Laboratories (3 mentions) Vorinostat, by LC Laboratories (2 mentions) Rpmi8226, by American Type Culture Collection (2 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Lonza (2 mentions) L glutamine, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Daudi, by American Type Culture Collection (2 mentions) Su dhl 4, by American Type Culture Collection (2 mentions) Ricolinostat, by Selleck Chemicals (2 mentions) Tubacin, by Selleck Chemicals (2 mentions) Entinostat, by LC Laboratories (2 mentions) Entinostat, by Selleck Chemicals (2 mentions) Compound c dihydrochloride, by R&D Systems (2 mentions) Belinostat, by Selleck Chemicals (2 mentions) Tunicamycin, by Enzo Life Sciences (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Caki 2, by American Type Culture Collection (1 mentions)

14 protocols using panobinostat

1

Renal Cancer Cell Line Sensitivity to Panobinostat and Nelfinavir

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Cell lines. Renal cancer cell lines (769-P, 786-O, Caki-2) were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were grown in Roswell Park Memorial Institute medium or McCoy's 5A medium supplemented with 10% fetal bovine serum and 1.0% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37˚C in a fully humidified atmosphere of 5% CO 2 .
Reagents. Panobinostat purchased from LC Laboratories (Boston, MA, USA) and nelfinavir purchased from Tocris Bioscience (Bristol, UK) were dissolved in dimethyl sulfoxide. Cycloheximide purchased from Enzo Life Sciences (Farmingdale, NY, USA) was dissolved in distilled water. All stock solutions were kept frozen at -20˚C until use.
Cell viability assay. Cells (5×10 3 ) were plated in a 96-well culture plate 1 day before treatment and then cultured for 48 hours in medium containing 15-60 nM Panobinostat with/without 10-20 μM nelfinavir. Cell viability was determined by MTS assay (CellTiter 96 Aqueous kit; Promega, Madison, WI, USA) following the manufacturer's instructions.
Okubo K., Isono M., Asano T, & Sato A. (2018). Panobinostat and Nelfinavir Inhibit Renal Cancer Growth by Inducing Endoplasmic Reticulum Stress. Anticancer research, 38(10).
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2

Cancer Drug Compound Preparation

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Erlotinib, pazopanib, everolimus, mocetinostat, vorinostat, panobinostat were purchased (LC Laboratories, Woburn, USA). Regorafenib (BAY 73–4506) was provided by Bayer Pharma AG Germany, EPZ011898–9 by Epizyme (Cambridge, MA, USA). Compounds were dissolved in dimethylsulfoxide (DMSO) and stored in 10 mmol/L stock solutions. Recombinant hEGF (Cell Signaling, Saint Quentin Yvelines, France), recombinant hFGF basic, and recombinant hKGF/FGF7 (R&D Systems, Lille, France) were dissolved in PBS.
Harttrampf A.C., da Costa M.E., Renoult A., Daudigeos-Dubus E, & Geoerger B. (2021). Histone deacetylase inhibitor panobinostat induces antitumor activity in epithelioid sarcoma and rhabdoid tumor by growth factor receptor modulation. BMC Cancer, 21, 833.
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3

HDACi Cytotoxicity Screening in CEFs

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CEFs (1 × 105) per condition were plated in duplicate in 12-well plates. The next day, cells were treated with different concentrations of HDACis ranging from nanomolar to millimolar concentrations for 48 h. HDACis that were used in this experiment were as follows: L-ascorbic acid (Sigma), trichostatin A (Sigma), valproic acid sodium salt (Sigma), sodium butyrate (Sigma), and panobinostat (LC Laboratories). Cell number and viability were determined with a Muse Cell Analyzer (Millipore), using the Muse Count and Viability dye per manufacturer's instructions.
Moshref M., Questa M., Lopez-Cervantes V., Sears T.K., Greathouse R.L., Crawford C.K, & Kol A. (2021). Panobinostat Effectively Increases Histone Acetylation and Alters Chromatin Accessibility Landscape in Canine Embryonic Fibroblasts but Does Not Enhance Cellular Reprogramming. Frontiers in Veterinary Science, 8, 716570.
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4

Optimizing Immune Modulation Experiments

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PMA (Sigma-Aldrich) was used at 0.4-100 ng/ml and ionomycin (Sigma-Aldrich) was used at a concentration of 100 ng/ml. 3-azido-3-deoxythymidine (zidovudine; AZT) (Sigma-Aldrich) was used at 10 µM, raltegravir at 5 µM and efavirenz at 0.32 µM were obtained from the NIH AIDS Research and Reference Reagent Program. TNFα (Merck) was used at a concentration of 0.08-2 ng/ml and LPS at 100 ng/ml (Merck). Fedratinib, TG101209, AZD1480, AZ960, gandolitinib, WP1066, XL109, ruxolitinib, momelotinib, JQ1 were used at a concentration of 0.2-5 µM, pacritinib at 40 nM-1 µM and Q-VD-Oph at 10 µM (all from Selleckchem). Vorinostat (SAHA) was used at a concentration of 0.2-5 µM (Prochifar srl, Italy) and panobinostat at 3.2-400 nM (LC Laboratories).
Ezeonwumelu I.J., García-Vidal E., Felip E., Puertas M.C., Oriol-Tordera B., Gutiérrez-Chamorro L., Gohr A., Ruiz-Riol M., Massanella M., Clotet B., Martinez-Picado J., Badia R., Riveira-Muñoz E, & Ballana E. (2022). IRF7 expression correlates with HIV latency reversal upon specific blockade of immune activation. Frontiers in Immunology, 13, 1001068.
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5

Generating Bortezomib-Refractory Myeloma and Lymphoma Cell Lines

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Multiple myeloma and lymphoma cell lines RPMI-8226, Kas6, Daudi, and SUDHL4, were from American Type Culture Collection (ATCC, Manassas, VA). To generate bortezomib-refractory cells, parental RPMI-8226 or Kas6 cells (designated as RMPI-8226wt or Kas6wt) were chronically exposed to increasing concentrations of bortezomib to generate resistant lines (designated as RMPI-8226v10r or Kas6v10r)[29 (link),30 (link)]. Resistant lines were then cultured in the presence of 10 nM bortezomib. Cell line identities were confirmed by Short Tandem Repeat DNA profiling as conducted by the MD Anderson Cancer Center Characterized Cell Line Core. All cell lines were cultured in RPMI1640 (Thermo Fisher Scientific, Waltham, MA) with 10% FBS (Thermo Fisher Scientific, Waltham, MA), 1% L-glutamine (Thermo Fisher Scientific, Waltham, MA), and 1% penicillin/streptomycin (Lonza, Basel, Switzerland) at 37 °C in a 5% CO2 incubator. All cultures were free of bacterial, fungal, and mycoplasma contamination (mycoplasma contamination was tested every 6 months). The following drugs were used for the study: bortezomib, LC Laboratories, (Woburn, MA); vorinostat, Cayman Chemical, (Ann Arbor, MI); panobinostat, LC Laboratories, (Woburn, MA); ricolinostat, Selleck Chemicals, (Houston, TX); tubacin, Selleck Chemicals, (Houston, TX).
Cheng T., Kiser K., Grasse L., Iles L., Bartholomeusz G., Samaniego F., Orlowski R.Z, & Chandra J. (2021). Expression of histone deacetylase (HDAC) family members in bortezomib-refractory multiple myeloma and modulation by panobinostat. Cancer Drug Resistance, 4(4), 888-902.
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6

Generating Bortezomib-Refractory Myeloma and Lymphoma Cell Lines

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Multiple myeloma and lymphoma cell lines RPMI-8226, Kas6, Daudi, and SUDHL4, were from American Type Culture Collection (ATCC, Manassas, VA). To generate bortezomib-refractory cells, parental RPMI-8226 or Kas6 cells (designated as RMPI-8226wt or Kas6wt) were chronically exposed to increasing concentrations of bortezomib to generate resistant lines (designated as RMPI-8226v10r or Kas6v10r)[29 (link),30 (link)]. Resistant lines were then cultured in the presence of 10 nM bortezomib. Cell line identities were confirmed by Short Tandem Repeat DNA profiling as conducted by the MD Anderson Cancer Center Characterized Cell Line Core. All cell lines were cultured in RPMI1640 (Thermo Fisher Scientific, Waltham, MA) with 10% FBS (Thermo Fisher Scientific, Waltham, MA), 1% L-glutamine (Thermo Fisher Scientific, Waltham, MA), and 1% penicillin/streptomycin (Lonza, Basel, Switzerland) at 37 °C in a 5% CO2 incubator. All cultures were free of bacterial, fungal, and mycoplasma contamination (mycoplasma contamination was tested every 6 months). The following drugs were used for the study: bortezomib, LC Laboratories, (Woburn, MA); vorinostat, Cayman Chemical, (Ann Arbor, MI); panobinostat, LC Laboratories, (Woburn, MA); ricolinostat, Selleck Chemicals, (Houston, TX); tubacin, Selleck Chemicals, (Houston, TX).
Cheng T., Kiser K., Grasse L., Iles L., Bartholomeusz G., Samaniego F., Orlowski R.Z, & Chandra J. (2021). Expression of histone deacetylase (HDAC) family members in bortezomib-refractory multiple myeloma and modulation by panobinostat. Cancer Drug Resistance, 4(4), 888-902.
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7

Cytotoxicity Assay for Ovarian Cancer

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Murine OVCA cell line ID8 and human OVCA cell lines A2780.IP2, A2780.CP20 (platinum resistant), and SKOV3.IP were plated in 96-well tissue culture plates (Corning Costar, NY) at 2000 cells/well in 45μl RPMI + 10% FBS. The plates were incubated at 37 °C in a humidified 5% CO2 atmosphere and treated on day 2 with a single dose of entinostat (LC Laboratories, Woburn, MA), panobinostat (LC Laboratories), azacytidine (TOCRIS, Bristol, UK) or entinostat combined with azacytidine. Testing was done in duplicate assays with 8 replicates each. Cells were collected after either 24 or 72 hours of drug exposure and cytotoxicity was evaluated using the ATPlite luminescence-based assay (PerkinElmer, Waltham, MA) as previously described [33 (link)].
Turner T.B., Meza-Perez S., Londoño A., Katre A., Peabody J.E., Smith H.J., Forero A., Norian L.A., Straughn JM J.r., Buchsbaum D.J., Rand all T.D, & Arend R.C. (2017). Epigenetic modifiers upregulate MHC II and impede ovarian cancer tumor growth. Oncotarget, 8(27), 44159-44170.
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8

Chromatin Immunoprecipitation Antibodies Protocol

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H3K9me3 antibody (Millipore, #07–442); HP1α antibody (cell signaling, #2616); Acetylated-Lysine (Ac-K2–100) antibody (Cell Signaling, #9814); Histone H3 antibody (Cell Signaling, #4499); Histone H4 antibody (Cell Signaling, #2935); H2AK5 antibody (Cell Signaling, #2576); H2BK5 antibody (Cell Signaling, #12799); H3K9Ac antibody (Cell Signaling, #9649); H3K14Ac antibody (Cell Signaling, #7627); H3K27Ac antibody (Cell Signaling, #8173); H3K56Ac antibody (Cell Signaling, #4243); H4K5Ac antibody (Cell Signaling, #8647); H4K8Ac antibody (Cell Signaling, #2594); H4K12Ac antibody (Cell Signaling, #2591); Anti-β-actin antibody (AC-15, Sigma); Anti-Hsp90 antibody (AC-16, Sigma) was used for western blot; 2-Deoxy-D-glucose (2-DG), bleomycin was brought from Sigma; HDAC inhibitor Panobinostat was purchased from LC Laboratories.
Liu X.S., Little J.B, & Yuan Z.M. (2015). Glycolytic metabolism influences global chromatin structure. Oncotarget, 6(6), 4214-4225.
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9

Solubilization of Pharmacological Agents

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Panobinostat purchased from LC Laboratories (Boston, MA, USA), suberoylanilide hydroxamic acid (SAHA) purchased from Cayman Chemical (Ann Arbor, MI, USA), entinostat purchased from Selleck Chemicals (Houston, TX, USA), and 5-aminoimidazole-4-carboxamide (AICAR) purchased from Sigma-Aldrich (St Louis, MO, USA) were dissolved in dimethyl sulfoxide. Metformin purchased from Enzo Life Sciences (Farmingdale, NY, USA) and compound C purchased from Selleck Chemicals were dissolved in distilled water. These agents were stored in the dark at −80 °C or− 20 °C until use.
Okubo K., Isono M., Asano T, & Sato A. (2019). Metformin Augments Panobinostat's Anti-Bladder Cancer Activity by Activating AMP-Activated Protein Kinase. Translational Oncology, 12(4), 669-682.
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10

Dissolution and Storage Protocols

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Vorinostat and fluvastatin purchased from Cayman Chemical, panobinostat purchased from LC Laboratories, belinostat purchased from Selleck Chemicals, and tunicamycin purchased from Enzo Life Sciences were dissolved in DMSO. Compound C dihydrochloride purchased from R&D Systems and cycloheximide purchased from Enzo Life Sciences were dissolved in distilled water. These reagents were stored at −80°C or −20°C until use.
Okubo K., Isono M., Miyai K., Asano T, & Sato A. (2019). Fluvastatin potentiates anticancer activity of vorinostat in renal cancer cells. Cancer Science, 111(1), 112-126.
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