Figure 3. Aerobic Glycolysis Is Required for Optimal IFN-γ Cytokine Production in T Cells.
(A) Intracellular IFN-γ was measured in T cells that were activated with anti-CD3/28 in medium containing either glucose (Glc) or galactose (Gal) for 4 days (top), or for 3 to 4 days in Glc followed by culture in Glc or Gal for 1 day (bottom, (B)–(H); or with oligomycin or rotenone/antimycin A for additional one day (C).
(B) IFN-γ in the culture supernatant was measured by ELISA after restimulation. Frequencies of IFN-γ-producing cells are shown (A) and (C).
(D) IFN-γ mRNA and T-bet expression.
(E) Western blot analysis of p-4EBP1 and p-S6K and p-AMPK and p-eIF2-α were examined.
(F) Hspa5 and Ppp1r15a expression after treatment with 0.1 mM thapsigargin (Thaps) for 4 hr.
(G) Mean cell diameter and volume as measured by laser light-scattering method.
(H) Total protein density (left) and total protein concentration (right) measured by Sypro Ruby staining and BCA assay, respectively.
Plots in (A) and (C) are representative of >3 independent experiments; (B) graph shows the mean ± SEM (*p = 0.001) from two independent experiments; (D) qPCR data are generated from five independent experiments and are shown as mean ± SEM, n.s., not significant, and the FACS plot is representative of two independent experiments; (E) blots are representative of two to four independent experiments; (F) results are presented as mean ± SEM from six independent experiments for cells without Thaps treatment and from one experiment for cells with Thaps treatment; (G) data are representative of two independent experiments; and (H) data are presented as mean ± SEM from three independent experiments. See also Figure S3.