Wild-type or plasmid-transfected HCC cells (500-1000/well) were seeded in six-well plates and treated with different doses of radiation (2.5, 5 and 10 Gy) following 24-hour pretreatment with pan-HDAC inhibitor (panobinostat [5 and 10 nM]; provide by Novartis Pharma AG, NIBR, Cambridge, MA, USA) or DMSO vehicle. The cells were cultured for 10 days, fixed, and stained with crystal violet. All colonies (clusters of more than 50 cells) visible to the naked eye were counted. The results were presented as means ± SEM of 3 independent experiments, with duplicate samples for each treatment condition.
Panobinostat
Manufactured by Novartis
Sourced in Switzerland, United States
Panobinostat is a histone deacetylase (HDAC) inhibitor. It is used as a lab research tool to study the effects of HDAC inhibition on cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Panobinostat, by Selleck Chemicals (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Wortmannin tlrl wtm, by InvivoGen (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Goat anti rabbit igg antibody, by Abcam (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Bafilomycin a1, by MedChemExpress (1 mentions)
Mirin, by MedChemExpress (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Vorinostat, by Selleck Chemicals (1 mentions)
Entinostat, by Selleck Chemicals (1 mentions)
Gsk126, by Active Motif (1 mentions)
Anti mouse ctla 4 clone 9h10, by BioXCell (1 mentions)
β actin ab, by Merck Group (1 mentions)
Anti mouse pd 1 clone rmp1 14, by BioXCell (1 mentions)
Actinomicin d, by Merck Group (1 mentions)
Anti mouse cd8 ab, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Corning (1 mentions)
Selumetinib, by Selleck Chemicals (1 mentions)
21 protocols using panobinostat
1
Radiosensitization of HCC Cells
2
Transgenic Rip1Tag2 Mouse Model
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The transgenic Rip1Tag2 mouse model (B6.D2-Tg(RIP1Tag2) 2Dh) has been previously described [17, 18] and was purchased from the NCI Mouse Repository. Mice were kept in a C57BL/6N background and maintained in a climate-controlled specific pathogen-free facility. Genotyping was done by PCR using the following primers (Biomers GmbH, Ulm): FW: 5′-GGACAAACCA-CAACTAGAATGCA-3′; RV: 5′-CAGAGCAGAATTGTG-GAGTG-3′. Rip1Tag2 is characterized by the transgenic expression of the simian virus 40 large T antigen (Tag) oncogene and the rat insulin promoter gene 1 (Rip1) that causes malignant transformation of β-cells [17] . Normal and hyperplastic islets of Rip1Tag2 mice have been identified with a diameter <400 µm, whereas insu- linomas must have a diameter >400 µm and show a well-marked angiogenesis [19] . Substances 100 pM bafilomycin A1 (Sigma-Aldrich, St. Louis, MO, USA), 1-100 nM panobinostat (Novartis AG, Basel, Switzerland), and 200 nM wortmannin (tlrl-wtm; InvivoGen, San Diego, CA, USA) were dissolved in sterile DMSO (WAK-Chemie Medical GmbH, Steinbach, Germany).
3
Evaluation of Anti-Cancer Combination Therapies
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Panobinostat was provided by Novartis Pharmaceuticals (Basel, Switzerland) and bortezomib was purchased from Millennium Pharmaceuticals (Cambridge, MA, USA). NVP-LDE225 was obtained from Selleck BioSciences Corporation (Shanghai, China) and chemotherapeutic drugs were purchased from Nanfang Hospital. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and anti-GAPDH and anti-β-actin antibodies were purchased from Sigma (St. Louis, MO, USA). An Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining kit was purchased from NanJing KeyGen Biotechnology (NanJing, China). Primary antibodies specific for phosphorylated IGF-1R (p-IGF-1R), IGF-1R, IRS-1, phosphorylated AKT (p-AKT), AKT, and Bcl-2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies specific for Sonic hedgehog (Shh), Ptch, Smo, and Gli-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Abcam (USA). An antibody specific for MRP1 and secondary horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG antibodies were obtained from Abcam (USA).
4
Investigating Chloroquine and NAC
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Chloroquine (CQ) and N-Acetylcysteine (NAC) were purchased from Sigma-Aldrich, Mirin and Bafilomycin A1 (Baf) were obtained from MedChemExpress, and Panobinostat (LBH) was provided by Novartis Pharmaceuticals.
5
Immunohistochemistry and Western Blot Analysis
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VPA was purchased from Enzo Life Sciences. Stock solutions were prepared in sterile water. Entinostat (MS-275) and vorinostat were from Selleck Chemicals; panobinostat (LBH589) from Novartis International; 5-Azacytidine (azacitidine) was from Sigma; GSK126 was from Active Biochem. Stock solutions were prepared in DMSO. Monoclonal antibodies anti-mouse PD-1 (clone RMP1-14, #BE0146) and anti-mouse CTLA-4 (clone 9H10, #BE0131) were purchased from Bioxcell. Actinomicin D was purchased from Sigma Aldrich.
All media, serum, antibiotics, and glutamine were from Corning.
Primary antibodies (Abs) for western blotting: β-Actin-Ab (Sigma-Aldrich, cod.A5316), Programmed death-ligand 1 (PD-L1)-Ab (Abcam, cod.Ab58810); (PD-L1)-Ab (cod.#13684), acetyl-H3-Ab (cod.#9649), PARP-Ab (cod.#9542) (Cell signaling Technology), and acetyl-H4-Ab (Millipore cod.06946). For IHC: monoclonal anti-mouse Ki67-Ab (Cell signaling Technology; cod.#12202), monoclonal anti-mouse CD4-Ab (Abcam, cod.Ab183685), anti-mouse CD8-Ab (eBioscience, clone 56-6.7). For immunofluorescence on fresh frozen tissues: anti-Foxp3-efluor570 (eBioscience clone FJK-16s, #41-5773-80).
All media, serum, antibiotics, and glutamine were from Corning.
Primary antibodies (Abs) for western blotting: β-Actin-Ab (Sigma-Aldrich, cod.A5316), Programmed death-ligand 1 (PD-L1)-Ab (Abcam, cod.Ab58810); (PD-L1)-Ab (cod.#13684), acetyl-H3-Ab (cod.#9649), PARP-Ab (cod.#9542) (Cell signaling Technology), and acetyl-H4-Ab (Millipore cod.06946). For IHC: monoclonal anti-mouse Ki67-Ab (Cell signaling Technology; cod.#12202), monoclonal anti-mouse CD4-Ab (Abcam, cod.Ab183685), anti-mouse CD8-Ab (eBioscience, clone 56-6.7). For immunofluorescence on fresh frozen tissues: anti-Foxp3-efluor570 (eBioscience clone FJK-16s, #41-5773-80).
6
Dissolution and Storage of Anticancer Compounds
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Selumetinib (AZD6244) was obtained from Selleck Chemicals (Houston, TX, USA). Sorafenib, p-Toluenesulfonate Salt was purchased from LC Laboratories (Woburn, MA, USA). Panobinostat was kindly provided from Novartis (Basel, Switzerland). All three compounds were dissolved in dimethylsulfoxide (DMSO) (WAK Chemicals; Steinbach, Germany) and stored at −20 °C.
7
Reagents for DNA Damage Repair Assays
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Carfilzomib was obtained from Selleck Chemicals. Pan-histone deacetylase (HDAC) inhibitors panobinostat (PS) and vorinostat (VS) were obtained from Novartis Pharmaceuticals (East Hanover, NJ) and Selleck Chemicals, respectively. All drugs were prepared as 10 mM stocks in 100% DMSO and stored in small aliquots at −80°C to prevent multiple free thaw cycles. Anti-phosphorylated (p)-ATR (S428), anti-p-CHK1(S345), anti-α/β tubulin, anti-DNA-PKcs, anti-acetyl histone H3 (K9/K14), anti-histone H3, anti-RAD52, anti-BRCA2 and anti-acetyl lysine antibodies were purchased from Cell Signaling Technology (Berverly, MA). Anti-hsp90α and anti-hsp70 antibody was purchased from Enzo Biosciences (Plymouth Meeting, PA). Anti-BRCA1 and anti-γ-H2AX antibodies were obtained from Millipore (Billerica, MA). Anti-CHK1, anti-ATR, anti-HDAC1, anti-HDAC2, and anti-HDAC3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-actin, anti-FLAG, anti-acetylated α-tubulin and anti-GFP antibodies and short hairpin RNAs against HDAC1, HDAC2 and HDAC3 were purchased from Sigma-Aldrich (St. Louis, MO). Acetylated-K69 hsp90 (Ac-K69 hsp90) antibody was previously described [24 (link)]. Anti-c-RAF antibody was purchased from BD Transduction Labs (San Jose, CA).
8
Investigating β-Catenin Antagonist BC2059
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β-Catenin antagonist, BC2059 was kindly provided by β-Cat Pharmaceuticals (Gaithersburg, MD). Panobinostat was kindly provided by Novartis Pharmaceuticals. Chemical structures are provided in Supplemental Figure 1 . All antibodies were obtained from commercial sources.
9
Investigating β-Catenin Antagonist BC2059
10
Osteosarcoma Cell Line Cultivation and Panobinostat Treatment
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Osteosarcoma cell lines Saos-2 (ACC243, Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Culture), MG63 (CRL-1427, ATCC, Manassas, USA) and U2-OS (kindly gift from Toh Weng Tan, Institute of Experimental Orthopaedics, Philipps University of Marburg) were grown in DMEM (Gibco, Paisley, UK) and MC3T3-E1 (ACC 210, DSMZ, Germany) mouse embryo osteoblasts were grown in MEM Alpha Medium (Gibco) both supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, penicillin (107 U/l) and streptomycin (10 mg/l) (Biochrom AG, Berlin Germany) at 37°C in a humidified atmosphere containing 5% CO2. hFOB 1.19 (CRL-11372, ATCC) were grown with 1:1 mixture of Ham's F12 Medium Dulbecco's Modified Eagle's Medium, with 2.5 mM L-glutamine, 0.3 mg/ml G418 and fetal bovine serum to a final concentration of 10% at 34°C to keep them undifferentiated. The temperature was raised up (39–40°C) to obtain a differentiated population that was also included in the study. Panobinostat, a kindly gift from Novartis AG, Basel Switzerland, was dissolved as previously described [12 (link)].
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