Figure 5. PEP Suppresses SERCA-Mediated ER Calcium Reuptake.

(A) Illustration of the glycolysis pathway and the targets of the indicated glycolytic inhibitors. 2-DG: 2-deoxyglucose; IAA: iodoacetate; OXA: oxalate.

(B) Heat map shows the normalized concentrations of the indicated glycolytic metabolites in CD4+ T cells stimulated with anti-CD3/anti-CD28 mAbs for 1 hr in the absence or presence of the glycolytic inhibitors described in (A) as measured by LC-QE-MS.

(C) Intracellular Ca²⁺ levels were measured in Fluo-4- and Fura-Red-labeled T_H1 cells treated with indicated glycolytic inhibitors before and after activation with ionomycin.

(D–F) T_H1 cells were left alone or stimulated with anti-CD3/anti-CD28 mAbs for 5 hr in 10 mM glucose in the absence or presence of indicated glycolytic inhibitors and the expression of CD40L (D), IFNγ (E) and TGFβ (Φ) was measured by flow cytometry (D) or ELISA (E and F).

(G and H) CD4⁺ T cells were transduced with empty vector control retroviruses (RV, Ctrl-KD) or those expressing enolase-1 shRNAi (eno1-KD). RV-infected T cells were left alone or stimulated with ionomycin for 10 min and the cytoplasmic versus nuclear distribution of NFAT1 was determined by Amnis Imagestream as described in Figure 3F (G), or alternatively were stimulated with anti-CD3/anti-CD28 mAbs in vitro in the presence of 10 mM glucose for 5 hr and the expression of IFNγ, IL-2 and CD40L was analyzed by flow cytometry (H).

(I) Intracellular Ca²⁺ levels in CD4⁺ T cells that were partially permeabilized and recovered in the absence or presence of PEP were measured as in (C) in the absence or presence of glycolytic inhibitor IAA.

(J) Ca²⁺-uptake assay as described in Figure 4F was performed on ER microsomal fractions isolated from Jurkat T cells cultured in the presence or absence of glucose or exogenous PEP. The addition of Tg served as a specificity control for SERCA-dependent activity.

Data shown are representative of two (H and J) and three (C and D, G and I) independent experiments or cumulative of three (B, E and F) independent experiments.

Data are expressed as mean ± SD and *p < 0.05 by unpaired Student's t test.