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Gsk126

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GSK126 is a potent and selective inhibitor of the EZH2 histone methyltransferase enzyme. It functions by blocking the catalytic activity of EZH2, which is involved in the regulation of gene expression through histone H3 lysine 27 trimethylation (H3K27me3).

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Lab products found in correlation

Diphenyl phosphine oxide 1 dpo 1, by Bio-Techne (2 mentions) 4 aminopyridine 4 ap, by Merck Group (2 mentions) Coulter counter analyzer, by Beckman Coulter (1 mentions) Hepg2, by American Type Culture Collection (1 mentions) Snu398, by American Type Culture Collection (1 mentions) Snu475, by American Type Culture Collection (1 mentions) Vorinostat, by Selleck Chemicals (1 mentions) Entinostat, by Selleck Chemicals (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) Panobinostat, by Novartis (1 mentions) Rabbit anti h3k27me3, by Cell Signaling Technology (1 mentions) Mouse anti ki67, by Cell Signaling Technology (1 mentions) Mouse anti carm1, by Cell Signaling Technology (1 mentions) Matrigel growth factor reduced basement membrane matrix, by Corning (1 mentions) Rabbit anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)

10 protocols using gsk126

1

Neuronal Excitability Modulation Assay

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4’Aminopyridine (4’AP) (50 µM, Sigma-Aldrich, St. Louis, MO, USA-Aldrich) was prepared in an aqueous solution and diphenyl phosphine oxide-1 (DPO-1) (310 nM, Tocris Bioscience, Bristol, UK) and GSK-126 (1 µM, 10 µM, Active Biochem, Maplewood, NJ, USA) were diluted in dimethyl sulfoxide (DMSO). Cells were pre-treated for 72 hours.
Ryland K.E., Svoboda L.K., Vesely E.D., McIntyre J.C., Zhang L., Martens J.R, & Lawlor E.R. (2014). Polycomb-Dependent Repression of the Potassium Channel-Encoding Gene KCNA5 Promotes Cancer Cell Survival Under Conditions of Stress. Oncogene, 34(35), 4591-4600.
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2

Cell Viability Assay with DAC and GSK-126

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SNU398, HepG2 and SNU475 cells were obtained from the American Type Culture Collection (ATCC; www.atcc.org). Each cell line was treated with daily doses of 100 nM DAC (Sigma-Aldrich) and/or 500 nM GSK-126 (Active Biochem, LTD, Hong Kong) for a total of three days. Treated cells continued to grow after the drug(s) were removed. SNU398 and HepG2 cells were harvested on day 14 after treatment, while SNU475 cells were harvested on day 24. Cell survival rates were assessed on specific days using a Coulter counter analyzer (Beckman Coulter, Inc.). Results are normalized to control group (cells treated with phosphate buffered saline (DPBS)). The data are presented as means of triplicate measurements; error bars represent standard errors of the mean (SEMs). The cell doubling times were calculated as described below: Doubling time = A*log(2)/(log(B) − log(C)), in which A is the number of days from cell seeded to harvested, B is the total number of harvested cells, and C is the number of seeded cells.
Zhang L., Li H.T., Shereda R., Lu Q., Weisenberger D.J., O’Connell C., Machida K., An W., Lenz H.J., El-Khoueiry A., Jones P.A., Liu M, & Liang G. (2022). DNMT and EZH2 inhibitors synergize to activate therapeutic targets in hepatocellular carcinoma. Cancer letters, 548, 215899.
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3

Antiviral Effects of GSK126 on VSV

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Cells were seeded into 24-well plates in DMEM 10% FBS. Once confluent, the cells were infected with VSV∆51-amiR-NTC or VSV∆51-amiR-4 by removing media, adding virus in serum-free media for 1 h, removing inoculum and adding supplemented media. Following 18-h incubation 15 µM GSK126 (Active Biochem) prepared in DMSO or equivalent volume of DMSO was added to the wells. The supernatant was collected for plaque assays, an alamarBlue® Assay was performed and cells were stained with crystal violet at the end of the experiment. A crystal violet cytotoxicity assay was then performed. See Supplementary Fig. 3a for the experimental timeline for different cell lines. For SEV transfer experiments, isolated SEVs derived from mock-infected cells or from MRBΔG-amiR-NTC- and MRBΔG-amiR-4-infected cells were transferred to a confluent monolayer of cells in 48-well plates. The treatment plan including SEV amount transferred can be found in Supplementary Fig. 4g.
Wedge M.E., Jennings V.A., Crupi M.J., Poutou J., Jamieson T., Pelin A., Pugliese G., de Souza C.T., Petryk J., Laight B.J., Boileau M., Taha Z., Alluqmani N., McKay H.E., Pikor L., Khan S.T., Azad T., Rezaei R., Austin B., He X., Mansfield D., Rose E., Brown E.E., Crawford N., Alkayyal A., Surendran A., Singaravelu R., Roy D.G., Migneco G., McSweeney B., Cottee M.L., Jacobus E.J., Keller B.A., Yamaguchi T.N., Boutros P.C., Geoffrion M., Rayner K.J., Chatterjee A., Auer R.C., Diallo J.S., Gibbings D., tenOever B.R., Melcher A., Bell J.C, & Ilkow C.S. (2022). Virally programmed extracellular vesicles sensitize cancer cells to oncolytic virus and small molecule therapy. Nature Communications, 13, 1898.
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4

Neuronal Excitability Modulation Assay

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4’Aminopyridine (4’AP) (50 µM, Sigma-Aldrich, St. Louis, MO, USA-Aldrich) was prepared in an aqueous solution and diphenyl phosphine oxide-1 (DPO-1) (310 nM, Tocris Bioscience, Bristol, UK) and GSK-126 (1 µM, 10 µM, Active Biochem, Maplewood, NJ, USA) were diluted in dimethyl sulfoxide (DMSO). Cells were pre-treated for 72 hours.
Ryland K.E., Svoboda L.K., Vesely E.D., McIntyre J.C., Zhang L., Martens J.R, & Lawlor E.R. (2014). Polycomb-Dependent Repression of the Potassium Channel-Encoding Gene KCNA5 Promotes Cancer Cell Survival Under Conditions of Stress. Oncogene, 34(35), 4591-4600.
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5

Epigenetic Modulation of Cancer Cell Lines

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SNU398, SNU475 and HepG2 cells were purchased form the American Type Culture Collection (ATCC; www.atcc.org), and treated with three daily consecutive doses of SGI-110 (Astex Pharmaceuticals, Dublin, CA) at 100 nM (72 h total exposure) or GSK126 (EZH2 inhibitor, Active Biochem, LTD, Hong Kong) at 500 nM daily. Cells were harvested at the time points indicated in Figure 2A.
Liu M., Zhang L., Li H., Hinoue T., Zhou W., Ohtani H., El-Khoueiry A., Daniels J., O’Connell C., Dorff T.B., Lu Q., Weisenberger D.J, & Liang G. (2018). Integrative Epigenetic Analysis Reveals Therapeutic Targets to the DNA Methyltransferase Inhibitor SGI-110 in Hepatocellular Carcinoma. Hepatology (Baltimore, Md.), 68(4), 1412-1428.
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6

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).
Terranova-Barberio M., Thomas S., Ali N., Pawlowska N., Park J., Krings G., Rosenblum M.D., Budillon A, & Munster P.N. (2017). HDAC inhibition potentiates immunotherapy in triple negative breast cancer. Oncotarget, 8(69), 114156-114172.
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7

Epigenetic Screening Protocol and Reagents

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Small molecules used in the epigenetic screen were obtained from Structural Genomics Consortium or The Wistar Institute Molecular Screening Facility. GSK126 was obtained from Active Biochem or Xcess Biosciences. Antibodies were obtained from: mouse anti-CARM1 (Cell Signaling, Cat. No: 12495, 1:1000 for immunoblotting), goat anti-BAF155 (Santa Cruz, Cat. No: SC9746, 1:1000 for immunoblotting), rabbit anti-methylated R1064 BAF155 (Millipore, Cat. No: ABE1339, 1:1000 for immunoblotting), rabbit anti-EZH2 (Cell Signaling, Cat. No: 5246, 1:1000 for immunoblotting), rabbit anti-cleaved PARP p85 (Promega, Cat. No: G7341, 1:1000 for immunoblotting), mouse anti-Ki67 (Cell Signaling, Cat. No: 9449, 1:500 for IHC), rabbit anti-cleaved caspase 3 (Cell Signaling, Cat. No: 9661, 1:1000 for immunoblotting and 1:50 for IHC), rabbit anti-H3K27Me3 (Cell Signaling, Cat. No: 9733, 1:1000 for immunoblotting and 1:100 for IHC), mouse anti-β-actin (Sigma, Cat. No: A1978, 1:20,000 for immunoblotting), rabbit anti-RNA pol II (Santa Cruz, Cat. No: sc-899). Growth factor reduced basement membrane matrix (Matrigel) was obtained from Corning. Unprocessed scans of blots are available in the Supplementary Fig. 7.
Karakashev S., Zhu H., Wu S., Yokoyama Y., Bitler B.G., Park P.H., Lee J.H., Kossenkov A.V., Gaonkar K.S., Yan H., Drapkin R., Conejo-Garcia J.R., Speicher D.W., Ordog T, & Zhang R. (2018). CARM1-expressing ovarian cancer depends on the histone methyltransferase EZH2 activity. Nature Communications, 9, 631.
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8

Ewing Sarcoma Cell Culture Protocol

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Ewing sarcoma cell lines were cultured in RPMI-1640 media (Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Atlas Biologicals, Inc., Fort Collins, CO, USA) and 6mM L-glutamine (Life Technologies, Grand Island, NY, USA) at 37°C and 5% CO2. For CHLA-25 cells, prior to cell seeding, plates were briefly coated (~5 minutes) with 0.2% Gelatin (Gelatin from bovine skin, Type B). For serum starvation conditions, cells were cultured as above without the presence of FBS for 24 hours. For hypoxia studies, cells were incubated inan xVivo system (Biospherix, Lacona, NY, USA) at 1% O2, 37°C and 5% CO2 for 48 hours. For GSK-126 studies, cells were treated with either vehicle control (DMSO; D128-500, Fisher Scientific, Waltham, MA) or 10μM GSK-126 (A-1275, Active Biochem, Maplewood, NJ) daily for 72 hours prior to functional studies.
Krook M.A., Hawkins A.G., Patel R.M., Lucas D.R., Van Noord R., Chugh R, & Lawlor E.R. (2016). A bivalent promoter contributes to stress-induced plasticity of CXCR4 in Ewing sarcoma. Oncotarget, 7(38), 61775-61788.
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9

Histone Purification and Western Blotting Protocol

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Core histones were purified using a commercial Histone Purification Kit (Active Motif, #40025, Carlsbad, CA, USA) following the manufacturer's instructions, with minor modifications. Briefly, HUVECs were treated with a potent and specific EZH2 inhibitor GSK126 (#A1275, Active Biochem, Hongkong) for the indicated time without changing the media. After washing twice with pre-warmed (37 °C) serum-free M200 media, 0.4 mL ice-cold Extraction Buffer was added to a 60 mm dish. Then, a plastic scraper was used to collect the cell protein extracts. The cells were rotated in Extraction Buffer overnight on a VWR rotating platform at 4 °C to release histones. The following day, cell extracts were transferred to fresh tubes and centrifuged in a microcentrifuge at maximum RCF for 5 min at 4 °C. Crude histones were neutralized with 0.1 mL 5X Neutralization Buffer and purified using columns provided. For Western blots, boiled histone lysates were separated by 12-15% SDS-PAGE. The same membrane was stripped and reprobed with anti-Histone 3 (H3) for equal loading.
Xu S., Xu Y., Yin M., Zhang S., Liu P., Koroleva M., Si S., Little P.J., Pelisek J, & Jin Z.G. (2018). Flow-dependent epigenetic regulation of IGFBP5 expression by H3K27me3 contributes to endothelial anti-inflammatory effects. Theranostics, 8(11), 3007-3021.
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10

Epigenetic Regulator GSK126 Treatment

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GSK126 was purchased from ActiveBiochem (Kowloon Bay, Kowloon, Hong Kong) and cells were treated with 5 µm for 48 h. MC3629 was synthesized as previously described [38].
Miele E., Po A., Mastronuzzi A., Carai A., Besharat Z.M., Pediconi N., Abballe L., Catanzaro G., Sabato C., De Smaele E., Canettieri G., Di Marcotullio L., Vacca A., Mai A., Levrero M., Pfister S.M., Kool M., Giangaspero F., Locatelli F, & Ferretti E. (2020). Downregulation of miR‐326 and its host gene β‐arrestin1 induces pro‐survival activity of E2F1 and promotes medulloblastoma growth. Molecular Oncology, 15(2), 523-542.
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