Mitochondria and TCR Signaling Figure 2. Metabolic changes elicited by mitochondrial drug treatments. (A) Relative concentrations of adenine nucleotides in 2C T cell extracts prepared after treatment with titrated concentrations of respective mitochondrial antagonists (75 min) were measured by HPLC analysis. Energy balance is defined as ratio of ([AMP] + [ADP]) to entire pool of adenine nucleotides, ([AMP] + [ADP] + [ATP]). Energy balances of drug-treated 2C T cells relative to that of DMSO-treated 2C T cells are plotted. Experiments were performed in duplicate. (B) Cell extracts prepared from 2C T cells after treatments with respective mitochondrial antagonists (75 min) were subjected to Western blotting (WB) for phospho-AMPKa1 (Thr172) and phospho-ACC (Ser79), respectively. WB analysis (re-blot) for AMPKa1 and ACC are also shown as loading controls. (C) Levels of intracellular superoxide (SO) of 2C T cells treated with respective mitochondrial antagonists, relative to that of 2C T cells treated with DMSO alone, are plotted. The SO level was measured by loading the cells with diethylethidium followed by flow cytometric analysis. Experiments in this Figure were repeated at least three times and the representative data are shown. doi:10.1371/journal.pone.0007738.g002 with Ld/QL9 complex is necessary and sufficient for near maximum level of F-actin polymerization and the costimulatory receptor/ligand interactions play minimal roles [15]. In striking contrast to the signaling events described above, the mitochondrial drug treatments, even at concentrations around the IC80s for the pMV-absorption, exerted little effects on the F-actin Figure 3. Inhibition by the mitochondrial drug treatments of activation of PLC-c1 and Akt and Ca2+ entry by L d B7-1ICAM-1 pMVs. (A and B) Cell extracts prepared from 2C T cells, pre-treated with titrated concentrations of respective mitochondrial antagonists (60 min) and then cultured for 15 min with peptide-loaded LdB7- 1ICAM-1 (A) or Ld (B) pMVs, were subjected to WB analysis for phospho- PLC-c1 (Tyr783) and phosph-Akt (Thr308), respectively. WB analysis (re- blot) for PLC-c1 and pan-Akt are also shown as loading controls. Refer to Figure S2 for relative intensities of bands of WB shown in B. (C) Ca2+ assays were performed with 2C T cells loaded with Indo-1. 2C T cells treated with the respective mitochondrial antagonists for 60 min were mixed with P1A (grey) or QL9 (black) peptide-loaded LdB7-1ICAM-1 pMVs (marked by arrows) and analyzed for changes in cytosolic [Ca2+] for 12 min. doi:10.1371/journal.pone.0007738.g003 PLoS ONE | www.plosone.org 4 November 2009 | Volume 4 | Issue 11 | e7738