Rik 2

For surface staining, cells were washed once with FACS buffer (PBS containing 0.1% BSA and 0.02% sodium azide) and cells were incubated for 15 min with the antibody mixture in FACS buffer. Cell surface staining was performed using the following antibodies: anti-CD3 (UCHT1, eBioscience™), anti-CD4 (RPA-T4, BioLegend, San Diego, USA), anti-CD8 (RPA-T8, BioLegend), anti-CD56 (HCD56, BioLegend), anti-HLA-DR (L243, BioLegend), anti-CD107a (H4A3, BioLegend), and anti-CD253/Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) (RIK-2, BD). The Fixable Viability Dye (FVD) eFluor™ 780 (eBioscience™) was used to exclude dead cells from the analysis. Cells were washed with FACS buffer and fixated with 4% PFA for up to 2 h. Cells were washed twice with Intracellular Staining Perm Wash Buffer (BioLegend) and incubated for 20 min with anti-GzmB (GB11, BD) and anti-IFNγ (XNG1.2, BD) in Intracellular Staining Perm Wash Buffer. Cells were washed again twice with Intracellular Staining Perm Wash Buffer, collected in FACS staining buffer, and stored at 4°C until acquisition. Samples were acquired with a BD LSR II flow cytometer with a HTS module and data were analyzed using FACSDiva and FlowJo Version 10.8 (both BD Becton Dickinson, Heidelberg, Germany).

After counting and culturing tumor cells in the appropriate wells, they were incubated with the drugs indicated. Cells were incubated with DCA (Sigma, St. Louis, MO, USA) at different concentrations, from 5–25 mM, for 24–72 h. Every 48 h, DCA was renewed. For the drug combination experiments, after 48 h or 72 h incubation with DCA, LUV-TRAIL (1000 ng/mL) was added for another 24 h before analysis of results. In some experiments, the general caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (abbreviated as z-VAD) (MedChemExpress, Monmouth Junction, NJ, USA) or the specific TRAIL blocking antibody RIK2 (BD Biosciences, Franklin Lakes, NJ, USA) were added to tumor cells.