N benzyloxycarbonyl val ala asp fluoromethylketone zvad fmk

EFM-192A, HeLa, MV4–11 and HT-29 cells were obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) and MDA-MB-231 and HEK 293 T cells from ATCC (American Type Culture Collection, CEM, Manassas, VA, USA). EFM-192A and MV4–11 cells were cultured in RPMI Medium 1640 GlutaMAX-I (Life Technologies, Inc., Eggenstein, Germany), HeLa, HEK 293 T and MDA-MB-231 cells in DMEM Medium (Life Technologies) and HT-29 cells were cultured in McCoy medium (Life Technologies), each supplemented with 10–20% fetal calf serum (FCS, Life Technologies), 1% penicillin/streptomycin (Life Technologies) and 1% sodium pyruvate (Life Technologies). Cell lines were authenticated by STR profiles and negatively tested for mycoplasma contamination. N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) was obtained from Bachem (Heidelberg, Germany), IFNβ from Biochrom (Ltd., Berlin, Germany), IFNγ from Merck & Co., Inc. (Darmstadt, Germany) and necrosulfonamide (NSA) from Calbiochem Merck & Co. Inc. The bivalent Smac mimetic BV6 was kindly provided by Genentech, Inc. (South San Francisco, CA, USA) [22] (link). All other chemicals were obtained from Sigma-Aldrich (Taufkirchen, Germany) or Carl Roth (Karlsruhe, Germany), unless otherwise indicated.

RMS cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) or the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) and cultivated in DMEM GlutaMAXX medium or RPMI 1640 medium (Life Technologies Inc., Eggenstein, Germany) with addition of 1% Penicillin/Streptomycin and 1 mM sodium pyruvate 10%-20% fetal calf serum (FCS), and incubated at 37°C with 5% CO₂ in air. Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coat from heathy blood donors by the Deutsches Rotes Kreuz Blutspendedienst Frankfurt. JQ1 was kindly provided by S. Knapp and M. Wanior (Frankfurt, Germany). The BH3 mimetics ABT-199, A-1331852, S63845 selectively targeting BCL-2, BCL-x_L, or MCL-1 were obtained from Selleck Chemicals (Houston, TX, USA) and the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) from Bachem (Heidelberg, Germany). All inhibitors were dissolved in dimethyl sulfoxide (DMSO) at variable stock concentrations.