Mitochondria and TCR Signaling polymerization. Thus, 2C T cells pre-treated (60 minutes) with the respective mitochondrial antagonists showed the comparable levels of F-actin content, when measured by flow cytometry after 15 minute culture with the QL9-loaded pMVs, as 2C T cells treated with DMSO alone (Figure 4A and C). In conformation of the importance of protein tyrosine kinase(s) (PTK) and phosphoinositide-3-kinase (PI3K) in membrane- proximal TCR signaling leading to the F-actin polymerization [30], both PP2 (a PTK inhibitor) and Wortmannin (a PI3K inhibitor) treatments strongly attenuated the F-actin polymeriza- tion (Figure 4B and C). Likewise, co-treatments of 2C T cells with PP2 (or Wotmannin) plus the respective mitochondrial antagonists also resulted in inhibition of the F-actin polymerization, supporting that the mitochondrial drug treatments had little effect on TCR-mediated PTK(s) and PI3K activation for the F-actin polymerization. Effects of the Mitochondrial Drug Treatments on Dynamic Rearrangement of F-Actin We also examined 2C T cells stained for F-actin under confocal microscope. As reported, 2C T cells cultured with QL9-loaded LdB7-1ICAM-1 pMVs showed enhanced cortical F-actin staining compared with control 2C T cells cultured with the P1A-loaded pMVs [15]. Consistent with results from flow cytometric analysis, 2C T cells, pre-treated with the respective mitochondrial drugs and then cultured with the QL9-loaded pMVs, also showed considerably higher levels of cortical F-actin staining than 2C T cells pre-treated with the same mitochondrial antagonist but Figure 4. Lack of inhibition by the mitochondrial drug treatments of F-actin polymerization by LdB7-1ICAM-1 pMVs. (A) 2C T cells pre- treated (60 min) with the respective mitochondrial antagonists (or DMSO) at concentrations near the IC80s for the pMV absorption (Deguelin; 750 nM, Atpenin; 3000 nM, Antimycin; 12.5 nM, FCCP; 2,500 nM, Phenformin; 200,000 nM) were cultured for 15 min with peptide-loaded LdB7-1ICAM-1 pMVs followed by F-actin staining and flow cytometric analysis. F-actin contents of 2C T cells, relative to that of 2C T ells treated with DMSO and cultured with the P1A-loaded pMVs, are plotted. (B) 2C T cells, pre-treated (60 min) with the respective mitochondrial antagonists (or DMSO) at concentration as in (A) along with either Wortmannin (0.2 mM) or PP2 (2 mM), were cultured for 15 min with the QL9-loaded pMVs followed by staining for F-actin. F- actin contents of 2C T cells co-treated with the respective mitochondrial antagonists plus Wortmannin (or PP2) are compared with that of 2C T cells treated with the respective mitochondrial antagonists alone. (C) Raw data obtained after treatments with DMSO alone, Deguelin and Antimycin, respectively, along with PP2 or Wortmannin are shown as representatives. doi:10.1371/journal.pone.0007738.g004 PLoS ONE | www.plosone.org 5 November 2009 | Volume 4 | Issue 11 | e7738