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Unc1999

Manufactured by Selleck Chemicals
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Sourced in United States, China, Germany

UNC1999 is a chemical compound developed for use in laboratory settings. It is a potent and selective inhibitor of the BET bromodomain proteins. UNC1999 is designed to facilitate research and investigation in the field of epigenetics and gene regulation.

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Lab products found in correlation

Gsk343, by Selleck Chemicals (6 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Alamar blue, by Thermo Fisher Scientific (2 mentions) Synergy neo hts, by Agilent Technologies (2 mentions) Egm 2 singlequot growth factor kit, by Lonza (2 mentions) Epz 5687, by Selleck Chemicals (2 mentions) Epz005687, by Selleck Chemicals (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Anti h3k27me3, by Cell Signaling Technology (2 mentions) Anti histone h3, by Merck Group (2 mentions) Anti arid1a, by Merck Group (2 mentions) Anti akt, by Cell Signaling Technology (2 mentions) Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions) Anti pik3ip1, by Santa Cruz Biotechnology (2 mentions) Anti pi3k p110alpha, by Cell Signaling Technology (2 mentions) Pbabe myr pik3ca143v, by Addgene (2 mentions) Anti arid1a, by Santa Cruz Biotechnology (2 mentions) Anti pakt t308, by Cell Signaling Technology (2 mentions) Anti ki67, by Cell Signaling Technology (2 mentions) Anti h3k9me3, by Abcam (2 mentions)

11 protocols using unc1999

1

Targeting CLL Cell Lines with Epigenetic Inhibitors

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CLL cell lines were treated with UNC1999 (S7164; Selleckchem), GSK343 (S7165; Selleckchem), and the PI3Kδ inhibitor idelalisib (GS-1101; Selleckchem). DZNep (120964–45-6) and JQ-(1268524–70-4) (Sigma Aldrich) treatments were incubated for 3 days using a different range of concentrations as mentioned in the figures. The levels of active AKT was analysed using the Akt Kinase Activity Assay Kit (ab139436; Abcam) according to the manufacturer’s instructions.
Kosalai S.T., Morsy M.H., Papakonstantinou N., Mansouri L., Stavroyianni N., Kanduri C., Stamatopoulos K., Rosenquist R, & Kanduri M. (2019). EZH2 upregulates the PI3K/AKT pathway through IGF1R and MYC in clinically aggressive chronic lymphocytic leukaemia. Epigenetics, 14(11), 1125-1140.
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2

Epigenetic Inhibitors in PDX Culture

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PDXs were dissociated as above. Single cell suspensions were filtered through 70 µm mesh, washed twice with wash buffer (PBS, 2% FBS and 1 mM EDTA) and red blood cells were lysed with ACK buffer (Crystalgen Inc.). Approximately 1×106 viable cells were seeded in 10 mL of DMEM:F12 supplemented with a EGM-2 SingleQuot growth factor kit (Lonza). Once cultures were established (~72 hours post-dissociation), 96-well plates were seeded with 5,000 cells/well in 100uL of ex vivo culture media and the following compounds were administered daily to a final concentration of 1uM: DMSO (vehicle), EPZ-5687, GSK343 or UNC1999 (Selleck Chemicals). Resazurin conversion of alamarBlue® (Invitrogen) was monitored on a compatible plate reader (Synergy Neo HTS; BioTek)
Poirier J.T., Gardner E.E., Connis N., Moreira A.L., de Stanchina E., Hann C.L, & Rudin C.M. (2015). DNA methylation in small cell lung cancer defines distinct disease subtypes and correlates with high expression of EZH2. Oncogene, 34(48), 5869-5878.
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3

Targeting Epigenetic Regulators in MCL

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Four MCL cell lines Granta-519, JVM-2, Mino, and Z138 were purchased from the ATCC (Manassas, VA, USA). Granta-519 was cultured with Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) and the rests were cultured with RPMI-1640 medium (Life Technologies, California, USA). Both media were supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin/streptomycin. HEK293T cells were routinely maintained in the lab and grown in DMEM supplemented with 10% FBS. All cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. Cells were plated at a density of 0.1 × 106 cells/flask in T25 flask for cell viability assay, at 0.5 × 106 cells/flask for apoptosis assays and cell cycle analysis, and at 1 × 106 cells/flask for Western blot. EPZ005687 and UNC1999 were purchased from Selleckchem (Houston, TX, USA). The compound was dissolved in anhydrous dimethyl sulfoxide (DMSO) to a stock solution of 10 mM, and then further diluted to the desired concentration for in vitro experiments.
Li W., Bi C., Han Y., Tian T., Wang X., Bao H., Xu X., Zhang X., Liu L., Zhang W., Gao H., Wang H., Zhang H., Meng B., Wang X, & Fu K. (2019). Targeting EZH1/2 induces cell cycle arrest and inhibits cell proliferation through reactivation of p57CDKN1C and TP53INP1 in mantle cell lymphoma. Cancer Biology & Medicine, 16(3), 530-541.
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4

Epigenetic Inhibitors in PDX Culture

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PDXs were dissociated as above. Single cell suspensions were filtered through 70 µm mesh, washed twice with wash buffer (PBS, 2% FBS and 1 mM EDTA) and red blood cells were lysed with ACK buffer (Crystalgen Inc.). Approximately 1×106 viable cells were seeded in 10 mL of DMEM:F12 supplemented with a EGM-2 SingleQuot growth factor kit (Lonza). Once cultures were established (~72 hours post-dissociation), 96-well plates were seeded with 5,000 cells/well in 100uL of ex vivo culture media and the following compounds were administered daily to a final concentration of 1uM: DMSO (vehicle), EPZ-5687, GSK343 or UNC1999 (Selleck Chemicals). Resazurin conversion of alamarBlue® (Invitrogen) was monitored on a compatible plate reader (Synergy Neo HTS; BioTek)
Poirier J.T., Gardner E.E., Connis N., Moreira A.L., de Stanchina E., Hann C.L, & Rudin C.M. (2015). DNA methylation in small cell lung cancer defines distinct disease subtypes and correlates with high expression of EZH2. Oncogene, 34(48), 5869-5878.
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5

Investigating EZH2 Inhibitor UNC1999 Effects

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The EZH2 inhibitor UNC1999 was purchased from Selleck Chemicals (Shanghai, China) and was dissolved in dimethyl sulfoxide (DMSO; concentrations used in experiments, 0.1, 1, 10 and 100 µM). Antibodies against phospho-JAK2 (1:1,000 dilution; cat. no. 3771S), JAK2 (1:1,000 dilution; cat. no. 3230S), phospho-STAT3 (1:1,000 dilution; cat. no. 9145S), STAT3 (1:1,000 dilution; cat. no. 9139S), EZH2 (1:1,000 dilution; cat. no. 5246S) and GAPDH (1:3,000 dilution; cat. no. 5174S), as well as horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG (1:2,000 dilution; cat. nos. 7074S and 7076S), were purchased from Cell Signaling Technology, Inc. (Boston, MA, USA).
Chen Z., Du Y., Liu X., Chen H., Weng X., Guo J., Wang M., Wang X, & Wang L. (2019). EZH2 inhibition suppresses bladder cancer cell growth and metastasis via the JAK2/STAT3 signaling pathway. Oncology Letters, 18(1), 907-915.
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6

HDAC Inhibitor Compound Synthesis

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UNC1999, GSK343, EPZ6438, GSK126 and Necrostatin were from Selleck Chemicals, Dexamethasone and Temozolomide from Sigma, Z-VAD-FMK from Calbiochem, and UNC2400, a negative control for UNC1999, was kindly provided by the SGC. The brain penetrant HDAC inhibitor Compound 26 was obtained from a six step synthesis starting from 4-bromo-2-nitroaniline based on a previously reported synthetic route [37 (link)]; dx.doi.org/10.1021/cn500021p).
Grinshtein N., Rioseco C.C., Marcellus R., Uehling D., Aman A., Lun X., Muto O., Podmore L., Lever J., Shen Y., Blough M.D., Cairncross G.J., Robbins S.M., Jones S.J., Marra M.A., Al-Awar R., Senger D.L, & Kaplan D.R. (2016). Small molecule epigenetic screen identifies novel EZH2 and HDAC inhibitors that target glioblastoma brain tumor-initiating cells. Oncotarget, 7(37), 59360-59376.
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7

Molecular Screening Assay Reagents

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Small molecules utilized in the screen were all obtained from the Molecular Screening Facility at The Wistar Institute. GSK126 was obtained from Xcess Biosciences and Active Biochem. UNC1999 was obtained from Selleckchem. The following antibodies from the indicated suppliers were used: anti-EZH2 (BD Bioscience, Cat. No: 612666, 1:1000), anti-EZH2 (Cell Signaling, Cat. No: 5246, 1:100), anti-ARID1A (Sigma, Cat. No: HPA005456, 1:1000), anti-H3K27Me3 (Cell Signaling, Cat. No: 9733, 1:1000), anti-β-actin (Sigma, Cat. No: A5441, 1:10,000), anti-ARID1A (Santa Cruz, Cat. No: sc-32761, 1:500), anti-Ki67 (Cell Signaling, Cat. No: 9449, 1:1000), anti-PIK3IP1 (Santa Cruz, Cat. No: sc-86785, 1:500), anti-Histone H3 (Millipore, Cat. No: 06-755, 1:1000), anti-GAPDH (Millipore, Cat. No: MAB374, 1:10,000), anti-cleaved caspase 3 (Cell Signaling, Cat. No: 9661, 1:10,000), anti-PI3K (p110alpha) (Cell Signaling, Cat. No: 4255, 1:1000), anti-pAKT (T308, Cell Signaling, Cat. No: 13038, 1:1000), anti-AKT (Cell Signaling, Cat. No: 9272, 1:1000) and anti-H3K9Me3 (Abcam, Cat. No: ab8898, 1:1000). pBabe-Myr-PIK3CA143V plasmid was obtained from Addgene. pBabe-EZH2, pBabe-EZH2 ΔSET and pQCXIP-PIK3IP1 plasmids were generated by standard molecular cloning protocols, and details are available upon request. Growth factor reduced Matrigel was purchased from BD Bioscience.
Biter B.G., Aird K.M., Garipov A., Li H., Amatangelo M., Kossenkov A.V., Schultz D.C., Liu Q., Shih I.M., Conejo-Garcia J.R., Speicher D.W, & Zhang R. (2015). Targeting EZH2 methyltransferase activity in ARID1A mutated cancer cells is synthetic lethal. Nature medicine, 21(3), 231-238.
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8

Molecular Screening Assay Reagents

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Small molecules utilized in the screen were all obtained from the Molecular Screening Facility at The Wistar Institute. GSK126 was obtained from Xcess Biosciences and Active Biochem. UNC1999 was obtained from Selleckchem. The following antibodies from the indicated suppliers were used: anti-EZH2 (BD Bioscience, Cat. No: 612666, 1:1000), anti-EZH2 (Cell Signaling, Cat. No: 5246, 1:100), anti-ARID1A (Sigma, Cat. No: HPA005456, 1:1000), anti-H3K27Me3 (Cell Signaling, Cat. No: 9733, 1:1000), anti-β-actin (Sigma, Cat. No: A5441, 1:10,000), anti-ARID1A (Santa Cruz, Cat. No: sc-32761, 1:500), anti-Ki67 (Cell Signaling, Cat. No: 9449, 1:1000), anti-PIK3IP1 (Santa Cruz, Cat. No: sc-86785, 1:500), anti-Histone H3 (Millipore, Cat. No: 06-755, 1:1000), anti-GAPDH (Millipore, Cat. No: MAB374, 1:10,000), anti-cleaved caspase 3 (Cell Signaling, Cat. No: 9661, 1:10,000), anti-PI3K (p110alpha) (Cell Signaling, Cat. No: 4255, 1:1000), anti-pAKT (T308, Cell Signaling, Cat. No: 13038, 1:1000), anti-AKT (Cell Signaling, Cat. No: 9272, 1:1000) and anti-H3K9Me3 (Abcam, Cat. No: ab8898, 1:1000). pBabe-Myr-PIK3CA143V plasmid was obtained from Addgene. pBabe-EZH2, pBabe-EZH2 ΔSET and pQCXIP-PIK3IP1 plasmids were generated by standard molecular cloning protocols, and details are available upon request. Growth factor reduced Matrigel was purchased from BD Bioscience.
Biter B.G., Aird K.M., Garipov A., Li H., Amatangelo M., Kossenkov A.V., Schultz D.C., Liu Q., Shih I.M., Conejo-Garcia J.R., Speicher D.W, & Zhang R. (2015). Targeting EZH2 methyltransferase activity in ARID1A mutated cancer cells is synthetic lethal. Nature medicine, 21(3), 231-238.
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9

Prostate Cancer Cell Line Manipulation

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The human prostate cancer cell lines 22RV1, DU145, LNcap, Vcap, and PC-3, as well as the human normal prostate epithelial cell line RWPE-1, were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). These cells were cultured in exactly matched basic medium containing 10% fetal bovine serum (Gibco) at 37 °C in standard humidified chambers with 5% CO2. Cell line identifies were confirmed by the short tandem repeat profiling method. To inhibit the pathways of methylation regulation, some inhibitors were used to treat prostate cancer cells with the indicated concentration and time. All these inhibitors were purchased from Selleck: GSK343 (S7164), gamma-Oryzanol (S3957), EED226 (S8496), CPI-455 HCl (S8287), and UNC1999 (S7165).
Huang K, & Tang Y. (2021). SChLAP1 promotes prostate cancer development through interacting with EZH2 to mediate promoter methylation modification of multiple miRNAs of chromosome 5 with a DNMT3a-feedback loop. Cell Death & Disease, 12(2), 188.
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10

Histone Modification Antibody Protocol

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UNC-0638 (cat no U4885) was from Sigma-Aldrich (Steinheim, Germany), UNC-1999 (cat no S7165) from Selleckchem (Houston, TX), H-Leu-Leu-OMe (LLME) (cat no 4000725.0005) from Bachem (Bubendorf, Switzerland). Nafamostat mesylate (cat no N0289), staurosporine (cat no S4400), mefloquine (cat no M2319), Pefabloc SC (cat no 11429868001), pepstatin A (cat no 516481) and E-64d (cat no E8640) were from Sigma-Aldrich (Steinheim, Germany). Rabbit anti-H3K9me2 antibody (cat no 07-441) was from EMD-Millipore (Darmstadt, Germany). Rabbit anti-H3K4me1 (cat no 5323S) monoclonal antibody was from Cell Signaling Technology (Danvers, MA). Rabbit anti-histone 2A (H2A) (cat no ab177308), H2B (cat no ab1790), H3 (cat no ab1791) and H4 (cat no ab177840) antibodies, mouse anti-H3K27me3 (cat no ab6002) monoclonal antibody and rabbit anti-H3S10p (cat no ab272166) polyclonal antibody were from Abcam (Cambridge, UK). A mouse monoclonal antibody to β-actin (cat no sc-517582) was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-H2BS14p polyclonal antibody (cat no PA5-105775) was from Invitrogen (Eugene, OR). Rabbit anti-H2BK16ac (cat no 39121) was from Active Motif (Carlsbad, CA).
Alanazi S., Rabelo Melo F, & Pejler G. (2021). Tryptase Regulates the Epigenetic Modification of Core Histones in Mast Cell Leukemia Cells. Frontiers in Immunology, 12, 804408.
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