Akti 1 2

T cells were cultured in RPMI medium (no glucose) containing 10% dialyzed serum (Thermo Fisher, Germany), 1 mM sodium pyruvate, 2 mM L-glutamine, with or without 10 mM glucose. For murine T cell activation, 96-well flat-bottom tissue culture plates were pre-coated with αCD3 antibody (5.0 μg/mL; eBioscience, Frankfurt, Germany), and cells were cultured with or without R837 (1 μg/mL)/R848 (10 μg/mL; InvivoGen, San Diego, CA, USA) for 24–48 h (5 × 10⁵ cells/well). For some experiments, cells were treated with the indicated inhibitors such as 2-DG (1 mM; Sigma, Germany), rapamycin (2 μM), and Akti-1/2 (1 μM; Sigma, Germany). For human T cell activation, 96-well flat-bottom tissue culture plates were pre-coated with αCD3 antibody (5.0 μg/mL; eBioscience, Frankfurt, Germany), and human CD8+ T cells were cultured with or without R848 (1 μg/mL; InvivoGen, San Diego, CA, USA) for 24 h (5 × 10⁵ cells/well). For the indicated experiments, human CD8+ T cells were treated with 2-DG (10 mM; Sigma, Germany), rapamycin (10 μM), and Akti-1/2 (5 μM; Sigma, Germany). For all experiments, triplicate wells were performed under each condition.

Cells were starved in serum-free α-minimal essential medium for 14–16 h before the experiment. Cells were then treated with indicated concentrations of inhibitors for 20 min: wortmannin (500 nM) and LY294002 (50 µM; Calbiochem, San Diego, CA, USA), KP372-1 (100 nM; Echelon Biosciences, Salt Lake City, UT, USA), and Akt-I-1/2 (100 nM; Sigma-Aldrich, St. Louis, MO, USA). ETOH and DMSO were used as vehicle controls. Inhibitors were delivered to each well in 1-µl volumes. After treatment with an inhibitor or vehicle control, cells were treated with 100 nM insulin (Novo Nordisk) or 5 µl H₂O for 30 min. Wells were then rinsed with 500 µl buffer A 3 times. Each well received 500 µl of transport buffer B and was incubated for exactly 10 min. Reactions were quenched by washing each well with 500 µl of ice-cold buffer A for P_i transport and buffer A + 5 mM d-glucose for glucose transport. Cells were lysed by treatment with 400 µl of 1 M NaOH and neutralized with 400 µl of 1 M HCl. In both assays, radioactivity was quantified by liquid scintillation counting, normalized to protein content determined by BCA protein assay (Thermo Fisher Scientific Life Sciences), and reported as moles of P_i or glucose transported per minute per mg protein.