Myc Influences Mitochondrial Dynamics Figure 3. Mitochondrial function. (a) Mitochondrial membrane potential. Cells were stained with JC-1 and analyzed by flow cytometry. The degree of mitochondrial membrane depolarization was measured by a decrease in the red/green fluorescence ratio. Values are expressed relative to those of myc+/+ cells, which were arbitrarily set to one. (b) OXPHOS. A Seahorse Bioscience XF24 Extracellular Flux Analyzer was used for the real-time determination of the oxygen consumption rate (OCR). Each inhibitor was injected at the times indicated by the arrows (injections: A-oligomycin, B- FCCP, C-2-DG, D-rotenone). A typical experiment, performed in triplicate wells is shown. The experiment was repeated on at least three occasions in replicates of four with similar results. When compared to myc2/2 cells, the p values for the basal OCR for myc+/+ cells = 0.000004 and for myc2/ 2wtMyc cells = 0.000001. (c) Steady-state ATP levels. The steady-state levels of ATP were measured for the myc+/+, myc2/2, and myc2/2wtMyc fibroblasts (left) and in MycER cells (right). The assays were performed both in the absence and presence of inhibitors of glycolysis (2-DG) and OXPHOS (rotenone) inhibitors. doi:10.1371/journal.pone.0037699.g003 containing a doxycycline (DOX)-inducible Myc-targeted shRNA (A549-shMyc cells). This vector also expressed red fluorescent protein, which permitted the convenient indirect monitoring of shRNA induction. As seen in Figure 4a, a four day DOX treatment of A549-shMyc cells led to the expression of red fluorescent protein, the abrupt cessation of proliferation, and a more flattened cellular morphology. Evaluation of these cells by immunoblotting showed these features to be associated with a nearly complete loss of endogenous Myc expression and a subsequent two-fold reduction in MitoTrackerH Green staining (Figure 4b and c). Metabolic analysis of these cells also revealed baseline levels of OXPHOS of DOX-treated A549-shMyc cells to be more than ten-fold reduced relative to non-DOX-treated cells (Figure 4d). Similar to what had been observed in myc2/2 cells, DOX-treated A549-shMyc cells displayed relatively low basal OXPHOS with little sRC, whereas cells grown in the absence of DOX showed higher levels of both basal OXPHOS and sRC. Collectively, these data indicate that Myc has a positive effect on mitochondrial mass and function in both rat and human cells. Regulation of mitochondrial ETC complexes by Myc The mitochondrial ETC consists of four major protein complexes that mediate a series of redox reactions involving electron transfer from donors (NADH, succinate and fatty acid oxidation products directly through coenzyme Q) to electroneg- ative acceptor molecules terminating with the reduction of oxygen to water [47]. The energy generated during this step-wise process is used to pump protons from the mitochondrial matrix to the inter-membrane space, thereby establishing an electrochemical proton gradient, which ultimately powers the conversion of ADP to ATP via ATP synthase (Complex V). Due to the large differences in the observed rates of OXPHOS and ATP biosynthesis in myc+/+, myc2/2, and myc2/2wtMyc cells (Figure 3), we compared the relative levels of their respective ETC complexes using blue native gel electrophoresis (BNGE) and scanning densitometry (Figure 5a and b). The identification of each complex was further confirmed by silver staining of SDS- PAGE gels to confirm the identity of individual subunits (not shown) and by in situ gel activity stains (see below) [48]. PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e37699