Extended Data Fig. 4: Bioenergetic changes induced by FOXK2 in 3T3-L1 adipocytes, FOXK2-induced 2-DG uptake under several inhibitor treatments, and role of FOXK1 and FOXK2 in T cell activation.

a, Glycolytic analysis of 3T3-L1 upon FOXK2 overexpression (FOXK2) or suppression (shFoxk2) compared to empty vector and shCtrl controls, respectively. Data are averages of four independent experiments, n = 12. b, Left, kinetic ECAR response of 3T3-L1 adipocytes overexpressing FOXK2 to glucose (10 mM), oligomycin (1 µM) and 2-DG (100 mM). A representative experiment out of three is shown, n = 3. Right, dissection of glycolysis and glycolytic capacity in 3T3-L1 adipocytes overexpressing FOXK2. Data are averages of four independent experiments, n = 4. The assay medium was substrate-free base medium supplemented with 2 mM glutamine. c, 2-DG uptake by 3T3-L1 adipocytes under basal conditions with overexpression of FOXK2 or an empty vector control after treatment with salirasib 25 μM for 2 h and rapamycin 100 nM for 16 h or 2 h. Representative experiment out of two is shown, n = 3. d–g, Human peripheral blood CD3⁺ T cells from 3 donors (n = 3) were transduced with Flag-tagged FOXK1 or FOXK2 vectors. Transduced cells were incubated in X-vivo 15 medium supplemented with 5% human serum and IL-2 for 3 days before Ki-67 staining and analysis by flow cytometry. Cells were gated on CD4 or CD8 and Flag-tag (FOXK1 or FOXK2 overexpression) before expression of the proliferation marker Ki-67 was analysed. Experiments were performed three times. Data shown as mean ± s.d. Unpaired two-sided Student’s t-test; ***P < 0.001, **P < 0.01, *P < 0.05.