Rvx 208

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.