Flow Cytometric Analysis of Intracellular Superoxide (SO) Purified 2C T cells (16105) in 100 ml of culture medium (DMEM) were treated with titrated concentrations of respective mitochondrial antagonists for 30 min at 37uC. Then, diethylethi- dum were added to the culture to 5 mM and incubated for 30 min at 37uC. The diethylethidium-treated 2C T cells were then stained with Alexa 647-conjugated anti-CD8 mAb for 10 min on ice, washed and resuspended in propidium iodide-containing FACS buffer (1X PBS, 10 mM HEPES, 2.5% horse serum, pH 7.2) and analyzed by flow cytometry. Flow Cytometric Analysis of F-Actin Content Purified 2C T cells (26105) in 100 ml of culture medium (DMEM medium) were treated with titrated concentration of respective mitochondrial antagonists for 60 min before culture with LdB7- 1ICAM-1 pMVs for 15 min. The 2C T cells were then fixed with paraformaldehyde (PFA) and stained with FITC-labeled Phalloidin and analyzed by flow cytometry as described previously [15]. Confocal Microscopy Purified 2C T cells (16105) in 100 ml of culture medium (DMEM medium) were treated with respective mitochondrial antagonists for 60 min before culture with LdB7-1ICAM-1 pMVs for 20 min on pre- warmed poly-L-lysine-coated coverslips. Then, the cells were fixed with PFA and stained with FITC-labeled Phalloidin and observed under the confocal microscope as described previously [15]. Western Blotting Analysis Preparation of cell extracts and Western blotting analysis were performed as described [15]. Calcium Assay Kinetic analysis of changes in cytosolic Ca2+ concentration was performed as described [50]. Briefly, 700 ml of purified CD8+ 2C T cells (1.46106) were loaded with 3 mM Indo-1/Am (Invitrogen) for 50 min at 37uC followed by thorough washing with ice-cold 1X PBS and resuspended in the culture medium (DMEM medium). Seven hundred microliters of Indo-1-loaded 2C T cells (1.56106) were treated with respective mitochondrial antagonists in DMSO (0.5% V/V) for 60 min in a 37uC water bath and mixed with 70 ml of peptide-loaded pMVs (1 mg/ml). Flow cytomeric analysis began 60 sec before addition of pMVs and continued for 720 sec afterward; FACS analysis was carried out with a LSR II flow cytometer equipped with a temperature-control device. Changes in the concentration of intracellular Ca2+ were determined measuring changes in the ratio of fluorescence intensities of Indo-1-loaded T cells detected by two separate photomultiplier tubes with bandpath filters of 440/40 (violet) and 530/30 (blue) nm, respectively; Indo-1 was excited by 351 nm UV laser. In Vitro T Cell Proliferation CFSE-labeled purified CD8+ 2C T cells (56104) in 90 ml of culture medium (RPMI) were cultured with 10 ml of LdB7- References 1. Smith-Garvin JE, Koretzky GA, Jordan MS (2009) T cell activation. Annu Rev Immunol 27: 591–619. 2. Pribila JT, Quale AC, Mueller KL, Shimizu Y (2004) Integrins and T cell- mediated immunity. Annu Rev Immunol 22: 157–180. 3. Vincenti F, Luggen M (2007) T cell costimulation: a rational target in the therapeutic armamentarium for autoimmune diseases and transplantation. Annu Rev Med 58: 347–358. 4. Mor A, Dustin ML, Philips MR (2007) Small GTPases and LFA-1 reciprocally modulate adhesion and signaling. Immunol Rev 218: 114–125. Mitochondria and TCR Signaling 1ICAM-1 Dros pMVs (0.5 mg/ml) loaded with either P1A or QL9 peptide in 96 well flat bottom plate in a 37uC humidified CO2 incubator. Extents of cell proliferation were determined by extents of CFSE dilution measured daily by flow cytometry [51]. At the beginning of the culture, 1 ml of respective mitochondrial antagonists in DMSO was added. Supporting Information Figure S1 HPLC analysis for adenine nucleotides. Adenine nucleotides, marked by arrows (black: AMP, yellow: ADP, red: ATP), in cell extracts were separated and quantitated by HPLC. Chromatograms obtained with cell extracts of 2C T cells treated with either DMSO or Deguelin at 300 nM and 750 nM, respectively, are shown as examples. Found at: doi:10.1371/journal.pone.0007738.s001 (0.03 MB PDF) Figure S2 Inhibition by the mitochondrial antagonists of activation of Akt and PLC-g1 by Ld pMVs X-ray films developed after WB analysis (Figure 3B) were scanned and intensities of each band relative to that of 2C T cells treated with DMSO alone and cultured with QL9-loaded Ld pMVs were calculated and plotted. Band intensity of each phospho-protein was compensated by that of the total protein in the corresponding sample. Found at: doi:10.1371/journal.pone.0007738.s002 (0.02 MB PDF) Figure S3 Effects of long-term mitochondrial drug treatments on viability and activation of 2C T cells cultured with peptide- loaded LdB7-1ICAM-1 pMVs. Purified CFSE-labeled CD8+ 2C T cells were cultured with P1A- or QL9-loaded LdB7-1ICAM-1 pMVs in the presence of the respective mitochondrial antagonists or DMSO alone as indicated for up to 3 days. Extents of cell proliferation judged by CFSE-dilution were analyzed daily by flow cytometry. Percentage of live cells and their mean FSC measured after 1 day of the culture were denoted inside the histograms as in Figure 6B. Found at: doi:10.1371/journal.pone.0007738.s003 (0.11 MB PDF) Acknowledgments We thank Mr. Alan Saluk, the director of Flow Cytometry Core Facility at TSRI, and his staffs for technical assistance. We thank Drs. Dale Boger and Martin Schnermann for providing us with synthetic Piericidin used in initial experiments. This is publication number 20334 from The Scripps Research Institute. Author Contributions Conceived and designed the experiments: IH. Performed the experiments: KK LW IH. Analyzed the data: KK LW IH. Wrote the paper: IH. 5. 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