Epz 6438

A375, FM28 and MCF7 cells were grown in RPMI (Sigma Aldrich, Brondby, Denmark), 10% FBS and penicillin/streptomycin. The FM28 melanoma cell line was originally established by A. Kirkin and kindly donated by Professor MH Andersen, Center for Cancer Immunotherapy (CCIT), Herlev Hospital, Denmark. Cell lines were kept at low passage and cultured for no more than 3 months. When relevant, cell identities according to ATCC were verified using DNA fingerprinting by short tandem repeat (STR) analysis (Cell IDTM system. Promega). Cell lines were frequently tested for mycoplasma (MycoAlert, Mycoplasma detection kit, Lonza). For blocking replication or mitosis prior to SSX2 expression, cells were treated with 10 μg/ml of aphidicolin (Sigma Aldrich) or 100 mM nocodazole (Sigma Aldrich), respectively, for two hours before addition of 100 ng/ml doxycycline. Replication was measured by incorporation of EdU using the Click-iT EdU imaging kit according to the manufacturer's recommendations (Thermo Fisher Scientific, Hvidovre, Denmark). The activity of EZH2 was blocked by addition of 10 μM EPZ6438 (Selleckchem, Munich, Germany).

For AlphaLISA, 10000 cells/well were seeded on a 384-well plate (#6005350, Perkin Elmer, Waltham, MA, USA) using the MultiFlo FX Microplate Dispenser. The next day, cells were treated with LPS (L9143-02, Sigma-Aldrich, Saint Louis, MO, USA) at a final concentration of 10 µg/mL, the plates were then incubated at 37 °C for 4 and 24 h, and the TNFa concentration was measured using AlphaLISA (Perkin Elmer, Waltham, MA, USA) on the Spark reader (Tecan, Männedorf, Switzerland) and the AlphaScreen module as per the manufacturer’s protocol. The CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) microplate assay was used to measure cell viability. Before the LPS challenge, cells were pre-treated for 1 h with the following drugs purchased from Selleckchem: AZD6244, SCH772984, MS-275, EX527, EPZ-5676, EPZ-6438, ORY-1001, RVX-208, OTX015, and (+)-JQ1 targeting the chromatin-associated proteins MEK 1/2, ERK 1/2, HDAC 1/2/3, SIRT1, DOT1L, EZH2, LSD1, BRD 2/3/4, BRD2/3, and BRD4 (Selleckchem, Houston, TX, USA), respectively, at the concentrations of 5, 1, 0.2, 0.04, 0.008, 0.0018, and 0.00032 µM. Drugs were dispensed using the Microlab STAR unit (Hamilton, Reno, NV, USA), while the transfer and media dilutions for the AlphaScreen readout were performed with the CyBio SELMA liquid handler (Analytic Jena, Jena, Germany). Measurements were performed in 12-plicate.

Human CRC cell lines HCT116 and SW620 were purchased from American Type Culture Collection. Cells were cultured in Dulbecco’s modified Eagle medium containing 10% fetal calf serum medium. All CRC cells were negative for mycoplasma contamination prior to use.
Oxaliplatin, GSK-J4, and EPZ-6438 were purchased from Selleck. DAPI (#5748) was obtained from R&D Systems. The following antibodies were used: H3K27me3 (9733, Cell Signaling Technology) for IHC, H3K27me3 (07–449; Millipore) for ChIP and western blot, H3K4me3 (61379, ActiveMotif), H3 (sc-10809, Santa Cruz), PAPR (9542, Cell Signaling Technology), γH2A.X (3322, Abways), and cleaved caspase 3 (9664, Cell Signaling Technology).

AZD7762 (CHK1 inhibitor), AZD6738 (ATR inhibitor), EPZ6438 (EZH2 inhibitor, also called Tazemetostat), KU-60019 (ATM inhibitor) were purchased from Selleckchem (Houston, TX, USA). GSK126 (an inhibitor of EZH2 methyltransferase activity) was purchased from Cayman Chemical (Ann Arbor, Michigan, USA). All drugs were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) and stored at -20 °C.