(A) ECAR, glycolysis, or maximal glycolytic capacity of CD8⁺ T cells transiently treated with vehicle (black), proteasome inhibitor (red), or proteasome activator (blue) prior to activation for 72 hours. Compounds were added at specified time points. (B) Glucose uptake, as measured by 2-NBDG, in CD8⁺ T cells treated as in A. (C) OCR and maximal respiratory capacity in CD8⁺ T cells transiently treated as in A. Compounds were added at specified time points. (D) mRNA expression of genes involved in glycolysis and glutaminolysis in vehicle- or proteasome inhibitor–treated CD8⁺ T cells. Expression was normalized to Actb. (E) Immunoblot analysis of Myc, HIF1α, Bcl-6, ERRα, and Foxo1 in vehicle-, proteasome inhibitor–, and proteasome activator–treated CD8⁺ T cells. The same samples were used to probe for both ERRα and Foxo1. Molecular weights in kDa are listed. (F) FACS analysis of IFN-γ production in CD8⁺ T cells transiently treated with proteasome inhibitor, expressed as fold change over vehicle, followed by addition of DMSO or Myc inhibitor at 24 hours after activation. (G–I) FACS analysis of IFN-γ production, expressed as fold change over WT, in (G) HIF1α-deficient, (H) Bcl-6–deficient, or (I) Foxo1-deficient CD8⁺ T cells transiently treated with proteasome inhibitor. (J) Myc expression (MFI) in first-division proteasome activity^loCD8^hi (red) and proteasome activity^hiCD8^lo (blue) cells. (K) Myc expression (MFI) in recently divided (second CFSE peak) cells arising from sorted undivided (first CFSE peak) CD8⁺ T cells treated with proteasome inhibitor or activator, relative to control-treated cells. Data are representative of 2 (E–I) or 3 independent experiments (A–D, J, and K), n ≥ 3 replicates per group. Error bars represent SEM of 3 replicates. *P < 0.05, **P < 0.01, ***P < 0.001 (A and C, 1-way ANOVA with Dunnett’s post-test; B, D, and F–K, Student’s 2-tailed t test).