Human pancreatic exocrine cells were either left untreated or exposed to 10 μM GSK126 (S7061, SelleckChem) or 1 μM Tazemetostat (S7128, SelleckChem) at a density of 1 × 106 cells per well for 24 h. After the initial 24 h incubation, fresh Miami Media was added, and the cells were cultured for an additional 24 h with either 10 μΜ GSK126 or 1 μM Tazemetostat. All incubations occurred in Miami Media 1 A (Mediatech/Corning 98-021, USA) supplemented with 2.5% human serum albumin (Australian Red Cross, Melbourne, VIC, Australia) in a cell culture incubator at 37 °C with 5% CO2 for a total of 48 h, using non-treated six-well culture plates (Corning). Because of low cell numbers isolated from the adult T1D donor, harvests were prioritised for gene expression and ChIP analyses, thus data for immunofluorescent staining and GSIS have not been provided.
Tazemetostat
Manufactured by Selleck Chemicals
Sourced in United States
Tazemetostat is a laboratory equipment product designed for scientific research. It functions as an inhibitor, specifically targeting the EZH2 enzyme. The core purpose of Tazemetostat is to facilitate the study of epigenetic regulation and its role in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Decitabine, by Selleck Chemicals (2 mentions)
Miami media 1a, by Corning (1 mentions)
Gsk126, by Selleck Chemicals (1 mentions)
Gapdh, by Merck Group (1 mentions)
Trametinib, by Chemietek (1 mentions)
Ab32158, by Abcam (1 mentions)
Ab19905, by Abcam (1 mentions)
Anacardic acid, by Selleck Chemicals (1 mentions)
Pinometostat, by Selleck Chemicals (1 mentions)
Gsk2879552, by Selleck Chemicals (1 mentions)
Quisinostat, by Selleck Chemicals (1 mentions)
Glutamax medium, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic anti anti, by Thermo Fisher Scientific (1 mentions)
Alpha mem glutamax, by Thermo Fisher Scientific (1 mentions)
Trypsin edta 1x, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Roswell park memorial institute (rpmi), by STEMCELL (1 mentions)
Gsk j4, by Merck Group (1 mentions)
10 protocols using tazemetostat
1
Epigenetic Regulation in Human Pancreatic Cells
2
Molecular Mechanisms of Epigenetic Regulation
3
Drug Dissolution and Dilution Protocol
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Quisinostat and Tazemetostat were purchased from Selleckchem (Houston, TX, USA). Drugs were dissolved in dimethyl sulfoxide (DMSO) to reach a stock concentration of 5 mM and diluted in the indicated fresh medium for in vitro studies.
4
SH-SY5Y Cell Treatment with Tazemetostat
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Treatment of SH-SY5Y cells with Tazemetostat (CAS No. 1403254–99-8, S7128, Selleckchem) was performed at 10 µM for 4 days starting from a 10 mM stock solution in DMSO; vehicle 0.1% DMSO was added to the controls.
5
Cell Line Cultivation and Epigenetic Inhibition
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hTERT‐HME1 cells were grown in Cascade Biologics Medium 171 (Gibco). MDA‐MB‐231 cells were grown in Leibovitz's (L‐15) + GlutaMAX Medium (Gibco) supplemented with 10% foetal bovine serum (FBS). MCF‐7 cells were grown in Minimum Essential Medium (MEM) Alpha + GlutaMAX (Gibco), supplemented with Mammary Epithelial Growth Supplement (MEGS) (Gibco, S0155), 10% FBS and 0.01mg/ml human recombinant insulin. All media were passed through vacuum filtration and were protected from contamination by adding 1X Anti‐Anti (antibiotic‐antimycotic) (Gibco). Cells were grown at 37°C at 5% CO2 in T‐75 and T‐150 vent flasks. Media was changed routinely, and cells were passaged using 0.25% Trypsin‐EDTA (1X) (Gibco). To respectively inhibit EZH2/1 or EZH2 methyltransferase function, 5μM UNC1999 (Cell Signaling Technology) or 5 μmol/L tazemetostat (Selleck Chemicals) were prepared in cell line‐specific media and administered to 80%‐90% confluent flasks for 48 hours. BAY11‐7082 inhibitor compound prepared in respective cell media (5 μmol/L) was administered for 24 hours to diminish NF‐κB activity. NKILA was inhibited by transfection of microRNA 103 using Lipofectamine RNAiMAX Reagent, per manufacturer protocol.
6
Myeloid Differentiation Induction Assay
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HL60 cells were cultured at 1,000,000 cells/mL in RPMI (StemCell, 36750) with 10% FBS (Sigma, F1051) with 1X Penicillin-Streptomycin (Gibco, Life Technologies, Fisher Thermo. 15140122) and 1X GlutaMAX (Gibco, Life Technologies, Fisher Thermo. 35050061) in the presence of 0.1uM ATRA (Sigma, R2625), 20uM Tazemetostat (Selleckchem, S7128) and 1uM GSK-J4 (Sigma, 420205) in 96-well V-shape bottom plate for 48 and 72 h. Concentration of DMSO control was adjusted to a maximum amount of DMSO (1.1%) used in treated wells. After 48 and 72 h, all cells were harvested, suspended in Hanks’ Balanced Salt Solution (HBSS; STEMCELL Technologies) supplemented with 2% FBS and 1.5μg/mL anti-human CD32 antibody (Clone IV.3; STEMCELL Technologies). Cells were stained for 20−30 min on ice with 1:200 anti-CD11b-BV711 (Clone M1/70; Biolegend) prior to analysis by flow cytometry. Flow cytometry data were analyzed in R using the package flowCore [73 ] and custom scripts.
7
Exploring Combination Therapies
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DOC (sanofi-aventis U.S. LLC, Bridgewater, NJ, USA), DZNep (Selleckchem, Houston, TX, USA), tazemetostat (Selleckchem), and human recombinant TRAIL (Sigma-Aldrich, St. Louis, MO, USA) were used at indicated concentrations.
8
In Vitro Epigenetic and Cytokine Treatment
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Decitabine (Sellekchem, Cat. No. S1200) in dimethyl sulfoxide (DMSO), tazemetostat (Sellekchem, Cat. No. S7128) in DMSO, and IFN-γ (R&D Systems, 285-IF-100/CF) in 1% FBS were administered in vitro using the same treatment schedule: cells were treated with noted concentrations (Decitabine 125-2000 nmol/mL, tazemetostat 312.5-5000 nmol/mL, IFN-γ 1-100 ng/mL) of each drug for 48 hours. Media was refreshed and new drug was added for an additional 48 hours. Untreated cells were given vehicle DMSO and media and were cultured for the duration of the drug treatment.
9
Compound Treatment on BCLs
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BCLs were continuously treated for 72 h in adherent conditions with bortezomib (stock concentration SC = [10 mM], Selleckchem), carfilzomib (SC = [10 mM], Selleckchem), chloroquine (SC = [10 mM], Selleckchem), Cortistatin A (SC = [100 μM], a kind gift from Prof. P. Baran, The Scripps Research Institute, La Jolla, CA, USA), docetaxel (SC = [10 mM], GSK343 (SC = [1 mM], Active Biochemicals Co), I‐CBP112 (SC = [10 mM], Sigma), JQ1 (SC = [10 mM], Selleckchem), mifepristone (SC = [10 mM], Selleckchem), Ro5‐3335 (SC = [10 mM], Calbiochem), ruxolitinib (SC = [10 mM], Selleckchem), SGC‐CBP30 (SC = [10 mM], Selleckchem), salinomycin (SC = [200 μM], Selleckchem), spliceostatin A (SC = [10 mM], Adooq Biosciences), tazemetostat (SC = [5 mM], Selleckchem), and 8‐AZA (SC = [100 mM], Chembiotech). All these compounds were resuspended in dimethyl sulfoxide (DMSO; Sigma), except for chloroquine resuspended in H2O. For the in vivo experiments, salinomycin (SC = [6 mg/ml], Medchemexpress) and JQ1 (SC = [100 mg/ml], Medchemexpress) were resuspended in a solution of DMSO/(2‐Hydroxypropyl)‐β‐cyclodextrin (HPCD) 10% (1:9, v/v).
10
Determining Cytotoxicity of Dasatinib and Tazemetostat
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Dasatinib and Tazemetostat (EPZ-6438) were obtained from Selleck Chemicals (Houston, 139 TX); To determine the IC50 of these drugs, lung adenocarcinoma cells were seeded in 140 triplicate at a density of 1,000 cells/well. The following day, cells were treated with the 141 drugs at increasing concentrations, and ATP lite assay (Promega) was performed after 72 142 hours of treatment. The dual drug studies (Dasatinib plus Tazemetostat) were performed 143 in a similar manner with the doses indicated in the figures. 144
For colony assay upon treatment with Dasatinib and Tazemetostat, cells were grown for 9 145 days with or without Tazemetostat (added every 3-4 days) at different indicated doses, 146 then they were seeded in triplicate at a density of 1,000 cells/well in a six-well multiwell. 147 Every 3-4 days, fresh medium was added with or without Dasatinib and Tazemetostat, 148 alone or in combination, at the indicated doses. Colonies were stained with crystal violet 149 and counted after 10-14 days. 150
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted July 11, 2020. ; https://doi.org/10.1101/2020.07.10.197392 doi: bioRxiv preprint
For colony assay upon treatment with Dasatinib and Tazemetostat, cells were grown for 9 145 days with or without Tazemetostat (added every 3-4 days) at different indicated doses, 146 then they were seeded in triplicate at a density of 1,000 cells/well in a six-well multiwell. 147 Every 3-4 days, fresh medium was added with or without Dasatinib and Tazemetostat, 148 alone or in combination, at the indicated doses. Colonies were stained with crystal violet 149 and counted after 10-14 days. 150
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted July 11, 2020. ; https://doi.org/10.1101/2020.07.10.197392 doi: bioRxiv preprint
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