Torin 1

NK cells were cultured in medium or with 200 U/ml IL-2 for 5, 10, 15, 20, or 24 h at 37°C and 5% CO₂ prior to stimulation. Where indicated, the following inhibitors were added to cells 1 hr prior to stimulation: 10 mM L-glutamic acid γ-(p-nitroanilide) hydrochloride (GPNA) (Santa Cruz Biotechnology) (1M GPNA solution was prepared in DMSO and kept at 37°C immediately prior to its addition), 100 mM D-phenylalanine (Sigma-Aldrich) (dissolved in medium for 1 h at 37°C by vortexing every 5-10 min prior to addition); 100 nM rapamycin (Calbiochem) (109.4 μM rapamycin in DMSO stock was stored at -20° C, thawed, and diluted in medium prior to addition); 100 nM Torin-1 (ApexBio) (1.645 mM Torin-1 in DMSO stock was stored at -80°C, thawed, and diluted in medium before addition). Nunc maxisorp ELISA plates (Thermo Fisher Scientific) were washed twice with PBS and then coated with 5 μg/ml anti-NKG2D mAb (1D11, BioLegend) or 5 μg/ml control mouse IgG₁ (MOPC-21, UCSF Monoclonal Antibody Core) in PBS for 24 h at 4°C. After coating, the plates were washed twice with PBS and blocked in complete medium for 10 min at room temperature (RT). For IFNγ measurements, GolgiSTOP was added to cells immediately prior to stimulation. Stimulation of 1.5 × 10⁵ cells/well proceeded for 5 h at 37°C and 5% CO₂.