Aeb071

In all experiments, cells were seeded at 2×10⁶ cells/mL and cultured in T cell media consisting of RPMI 1640 supplemented with 10% FBS, 10mM Hepes (pH 7.0), 2mM L-glutamine, 1mM sodium pyruvate, 1x nonessential amino acids, penicillin, streptomycin (all from ThermoFisher, Waltham, MA, USA), and 50μM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). For flow cytometry, 2×10⁶ splenocytes were used per sample. For all other assays 10×10⁶ sorted T cells were used. Cells were stimulated with either 1ug/mL anti-CD3 antibodies (BD Biosciences), 10μg/mL PMA, 1μM thapsigargin (ThermoFisher), or 250ng/mL Ionomycin (Sigma) for the times indicated. In some experiments, cells were pretreated for 30 minutes with the following inhibitors: 10nM Staurosporine, 1μM CID2858522, 100nM 6-Amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) (all from MilliporeSigma, Burlington, MA, USA), 10μM AEB071, 5μM SB203580, 0.5μM BIRB796 (all from Selleckchem, Houston, TX, USA), 10μM U0126 (Cell Signaling Technology, Danvers, MA, USA), 10μM BAPTA-AM (ThermoFisher), or DMSO (vehicle) and then activated with either 1ug/mL anti-CD3 antibodies or 10μg/mL PMA for the times indicated. For qRT-PCR experiments, following activation, samples were split in half, with one half (5×10⁶ cells) receiving 1000 U/mL recombinant human IL-2 and the other half receiving no further treatment.