Otx015

TNBC and ovarian cell lines, MDA-MB-231 and BT549 and SKOV3, respectively, were cultured in DMEM, and ovarian cells OVCAR3 were cultured in RPMI supplemented with inactivated fetal bovine serum (10%), antibiotics (100 U/mL penicillin and 100 /mL streptomycin) and L-glutamine (2 mM) (Gibco (Thermofisher), Sigma-Aldrich) (37 °C, 5% CO₂). All cell lines used were provided by Drs. J. Losada and A. Balmain, who purchased them from the ATCC, in 2015. Cells authenticity was confirmed by STR analysis at the molecular biology unit at the Salamanca University Hospital. MDA-MB-231-derived resistant cell line (MDA-MB-231R) was obtained by pulsed exposure to increasing doses of JQ1 (72 h pulses every 2 weeks for 6 months).
BET inhibitors (JQ1 (HPLC: 99.6% purity) and OTX-015 (HPLC: 99.82% purity) and PROTACs-BRD4 (MZ1 (HPLC: 99.5% purity) and ARV-825 (LCMS: 99.37% purity)), together with the inactive form of MZ1, cis-MZ1 (HPLC: 98.6% purity), were purchase from Selleckchem (Houston, TX) and Tocris Bioscience (Bio-Techne R&D Systems, S.LU).

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.

ABT263, JQ1 and OTX015 were purchased from Selleckchem (Houston, TX). A 10 mM working solution in dimethylsulfoxide (DMSO) was prepared for all reagents prior to storage at -20°C. Final concentrations of DMSO were below 0.1% (v/v).