PLC/PRF/5, HepG2, and Huh7 cells were maintained in a humidified incubator at 37 °C in an atmosphere of 5% CO2. PLC/PRF/5 cells were cultured in RPMI-1640 medium (Welgene, Gyeongsan, Korea) supplemented with 10% (v/v) fetal bovine serum (FBS; Atlas Biologicals, Fort Collins, CO, USA) and 1× penicillin-streptomycin solution (Cat. #30–002-cl; Corning, Mediatech Inc., Manassas, VA, USA). HepG2 and Huh7 cells were cultured in DMEM (Welgene) containing 10% (v/v) FBS (Atlas Biologicals) and 1× penicillin-streptomycin solution (Cat. #30–002-cl; Corning, Mediatech Inc.). For drug treatment, JIB-04 (Cat. #15338; Cayman Chemical, Ann Arbor, MI, USA) and trichostatin A (TSA; Cayman Chemical, Ann Arbor, MI, USA) were individually dissolved in dimethyl sulfoxide (DMSO) and diluted before use. The PLC/PRF/5 cell line was provided kindly by Dr. Injae Shin (Yonsei University, Seoul, Korea), and the HepG2 and Huh7 cell lines by Dr. Wang Shick Ryu (Yonsei University, Korea). All cell lines were tested for mycoplasma contamination.
Trichostatin a tsa
Manufactured by Cayman Chemical
Sourced in United States
Trichostatin A (TSA) is a histone deacetylase (HDAC) inhibitor. It is a small molecule that modulates the activity of HDAC enzymes, which play a role in regulating gene expression. TSA can be used as a research tool to investigate the biological functions of HDAC enzymes and their influence on cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sodium butyrate, by Merck Group (2 mentions)
Ly294002, by Cayman Chemical (2 mentions)
5 aza 2 deoxycytidine, by Merck Group (2 mentions)
5 aza 2 deoxycytidine aza, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Welgene (1 mentions)
Fetal bovine serum (fbs), by Atlas Biologicals (1 mentions)
Rpmi 1640 medium, by Welgene (1 mentions)
Ve822, by Cayman Chemical (1 mentions)
Mirin, by Cayman Chemical (1 mentions)
Azd7762, by Cayman Chemical (1 mentions)
Nu7026, by Cayman Chemical (1 mentions)
Nu7441, by Bio-Techne (1 mentions)
5 aza 2 deoxycytidine 5 aza, by Merck Group (1 mentions)
Taxol, by Merck Group (1 mentions)
Suberoylanilide hydroxamic acid, by Cayman Chemical (1 mentions)
Rpmi 1640 medium, by Corning (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Ly2109761, by Cayman Chemical (1 mentions)
23 protocols using trichostatin a tsa
1
Culturing Hepatocellular Carcinoma Cell Lines
2
Comprehensive Small Molecule Library for DNA Repair
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Commercially available small molecules used in this study were SCR7 (Tocris; 5342), Azidothymidine (AZT; Tocris), B02 (Sigma; SML0364), 6-Hydroxy-DL-DOPA (DOPA; Tocris), Cyclosporin H (CsH) (Sigma; SML 1575), Mirin (Cayman; 13208), Olaparib (LC Labs; O-9201), AZD7762 (Cayman; 11491), VE822 (Cayman; 24198), RS1 (Calbiochem; 553510), NU7026 (Cayman; 13308), NU7441 (Tocris; 3712), Trichostatin A (TSA) (Cayman; 89730), Pevonedistat (MLN4924) (Selleckchem; S7109), and M3814 (MedKoo; 206478). As a control, this study included the CRISPY Mix (51 (link)), which is the combination of 20 μM NU7026 (NHEJ inhibitor), 0.01 μM TSA (HDAC inhibitor) and 0.5 μM MLN4924 (improves CtIP) (NSC 15520 was not available). Stock solutions were prepared in dimethylsulfoxide (DMSO) (Sigma) and diluted to working concentrations before use. The medium was changed 24 h after the addition of small molecules. We listed the detailed information and working concentrations of small molecules in Supplementary Table S2 .
3
Silenced JPH3 Epigenetic Regulation
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Cells with silenced JPH3 expression were seeded at a density of 1×106 cells/ml. After overnight culture, cells were treated with the demethylation agent 5-aza-2'-deoxycytidine (5-Aza; Sigma-Aldrich, St Louis, MO) at a final concentration of 10 µM for 72 hours and then further treated with 100 nM trichostatin A (TSA; Cayman Chemical Co, Ann Arbor, MI) for 16 hours. Cells were subsequently harvested for DNA and RNA extraction.
4
Establishment of Lung Adenocarcinoma Cell Models
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Human lung adenocarcinoma epithelial cell lines, A549 and H1299, were cultured with RPMI 1640 medium (Corning, New York, NY, USA) containing 10% fetal bovine serum (FBS) (Gibco, Invitrogen, NY, USA), 100 μg/ml streptomycin and 100 U/ml penicillin G. A549-T24 cells were obtained by exposing A549 cells to stepwise increased dose of Taxol until 24 nM (Sigma Aldrich, St. Louis, MO, USA). E2-A549 cells were obtained by treating A549 cells with 1 μg/ml estradiol (E2) for 3 weeks. All cells were maintained in a humidified incubator under 5% CO2 at 37 °C. Trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) were purchased from Cayman Chemical (Ann Arbor, MI, USA).
5
HDAC3 Inhibition and IP4 Signaling
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Trichostatin A (TSA) and D-myo-inositol-1,4,5,6-tetraphosphate (IP4(1,4,5,6)) were obtained from Cayman. The anti-HDAC3-N′ N-19 antibody was from Santa Cruz Biotechnology. Anti-FLAG M2-agarose and anti-FLAG antibody were from MilliporeSigma.
6
Immunoblotting for EMT and Stemness Markers
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For immunoblotting, antibodies against STAT1 (#9172), phospho-STAT1 (#9171), Smad2/3 (#5678), and phospho-Smad2 (#3108) were acquired from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin (sc-4778), -Klf4 (sc-166229), -HDAC4 (sc-5245), and -Sp1 (sc-17824) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and antibodies against E-cadherin (ab15148), N-cadherin (ab18203), vimentin (ab137321), Snail (ab180714), Twist (ab175430), Bmi1 (ab126783), Sox2 (ab97959), and Oct4 (ab109183) were obtained from Abcam (Cambridge, MA, USA). LY2109761 and trichostatin A (TSA) were purchased from Cayman Chemical (Ann Arbor, MI, USA) and Sigma-Aldrich/Merck KGaA, respectively. The STAT1-shRNA vector was acquired from Origene (Rockville, MD, USA).
7
Molecular Mechanisms of Cellular Regulation
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TPA and HRP-conjugated goat anti-rabbit (A6154) and anti-mouse (A4416) IgG were purchased from Sigma-Aldrich Co. (St. Louis, MO). A PKC inhibitor (GF109203X) and actinomycin D (ActD) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Cyclopentylidene-(4-(4′-chlorophenyl)thiazol-2-yl)hydrazone (CPTH2) was purchased from Calbiochem (San Diego, CA). 5-(and-6)-Carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFHDA) and dihydroethidium (DHE) were purchased from Molecular Probes (Eugene, OR). Diphenyleneiodonium (DPI) and garcinol (Gar) were purchased from Enzo Life Sciences Inc. (Farmingdale, NY). Trichostatin A (TSA) was purchased from Cayman Chemical (Ann Arbor, MI). An anti-phospho-PKC (pan) (βII Ser660) rabbit polyclonal antibody (#9371) and normal rabbit IgG (#2729) were purchased from Cell Signaling Technology (Danvers, MA). Anti-actin mouse monoclonal antibody (MAB1501) and anti-acetyl-histone H3 (#06-599) and H4 (#06-598) rabbit polyclonal antibodies were purchased from Millipore Co. (Billerica, MA).
8
Molecular Modulation of Cellular Dynamics
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For actin depolymerization studies, Cytochalasin D (cat # 11330, Caymen Chemical) was used at 10 μg/mL for 1 h. To inhibit Rho A kinase, 50 μM Y-27632 (cat #72302, Stem Cell Technologies) was used for 1 h prior to FRET imaging to reduce myosin activity. For EMT induction, recombinant human TGF-β1 (R&D systems) was used to induce EMT at a concentration of 2 ng/mL for 24 h. Modifications in DNA ultrastructure were done to condense or decondense chromatin with the use of 600 nM trichostatin A (TSA) for 4 h (Cayman Chemical Company), to increase euchromatin, and 2.5 μM methylstat (Sigma Aldrich) for 48 h, to increase heterochromatin. For the cell cycle synchronization assay, Aphidicolin (cat #57-361, Thermo Fisher Scientific) was used to block the cells in early S-phase, at 3 µg/mL for 24 h.
9
Cell Line and Primary Hepatocyte Cultivation
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The HCC cell lines HepG2 (ATCC HB-8065), PLC (ATCC CRL-8024), Hep3B (ATCC HB-8064) and Huh7 were cultured as described [53 (link)]. Primary human hepatocytes were isolated by the Biobank of the Department of General, Visceral and Transplant Surgery in Ludwig-Maximilians University using a two-step collagenase perfusion technique with modifications [54 (link)].
The murine Hepa129 cell line originates from a C3H/HeN mouse and was obtained from the NCI-Frederick Cancer Research and Development Center (DCT Tumor Repository). The murine Hepa1-6 cell line (ATCC CRL-1830) was also used. Isolation and culture of primary murine hepatocytes (PMH) were performed as described [55 (link)].
For stimulation experiments, cells were treated with trichostatin A (TSA) (Cayman Chemical, Ann Arbor, MI, USA), suberoylanilide hydroxamic acid (SAHA) (Cayman Chemical) and sorafenib (Biovision, Milpitas, CA, USA) at the concentrations and for duration as indicated.
The murine Hepa129 cell line originates from a C3H/HeN mouse and was obtained from the NCI-Frederick Cancer Research and Development Center (DCT Tumor Repository). The murine Hepa1-6 cell line (ATCC CRL-1830) was also used. Isolation and culture of primary murine hepatocytes (PMH) were performed as described [55 (link)].
For stimulation experiments, cells were treated with trichostatin A (TSA) (Cayman Chemical, Ann Arbor, MI, USA), suberoylanilide hydroxamic acid (SAHA) (Cayman Chemical) and sorafenib (Biovision, Milpitas, CA, USA) at the concentrations and for duration as indicated.
10
Cell Culture Media and Supplements
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Cell culture media and supplements such as RPMI 1640, DMEM, PBS, and antibiotics (penicillin, streptomycin, and puromycin) were purchased from Lonza (Allendale, NJ), Fisher Scientific (Pittsburgh, PA) or Santa Cruz Biotechnology (Dallas, TX). Heat-inactivated fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Flowery Branch, GA). Sodium butyrate and sanguinarine were procured from Sigma-Aldrich (St. Louis, MO). Trichostatin A (TSA), apicidin, HC toxin, LY294002, PX866, CAL-101, MK2206, Triciribine, GDC0068, Rapamycin, AZD8055, and BEZ235 were obtained from Cayman Chemical (Ann Arbor, MI). Tetrandrine was acquired from Santa Cruz Biotechnology, and (–)-depudecin was purchased from BioVision (Milpitas, CA) and MyBioSource (San Diego, CA). Datiscetin was ordered from BOC Sciences (Shirley, NY), while wortmannin and CUDC-907 were procured from Selleck Chemicals (Houston, TX).
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