[22,24,25,32,56]. Higher levels of fission proteins might be needed to maintain homeostasis of the greatly increased mitochondrial mass in Myc overexpressing cells as well as to provide a means for the more rapid elimination of oxidatively damaged organelles that are generated as a result of high levels of reactive oxygen species induced by Myc [26,27]. Our results suggest that the increased mitochondrial mass in response to Myc induction may be associated with high levels of organelle turnover, thus necessitating higher levels of both fission and fusion proteins. In conclusion, we propose that Myc, which is among the first genes activated in response to a broad array of growth factors, serves to link the proliferative signals mediated by these factors with mitochondrial biogenesis and function via the control of fission and fusion processes [32]. That Myc appears capable of independently controlling both of these arms, coupled with its rapid induction by growth factors and its extremely short half-life, provides the means by which mitochondrial supply and cellular energy generation can be fine tuned to meet cellular proliferative demands. Materials and Methods Cell culture and virus infections The generation of myc+/+ and myc2/2 rat fibroblasts has been previously described [33,34]. myc2/2wtMyc cells were generated by stable transduction with a lentiviral vector expressing a full- length human wtMyc as previously described [43], and cells were selected for blasticidin resistance. MycER cells were also derived from myc2/2 cells by transduction with the pBabePuro-MycER retroviral vector and selection in puromycin [35]. To activate MycER, 4-HT (Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 250 nM and the media was replaced every 48–72 hours for all experiments. DsRed and GFP, both fused to the mitochondrial localization signal of subunit VIII of cytochrome c oxidase (pMito-DsRed and pMito-GFP, Clontech, Mountain View CA) were amplified using the polymerase chain reaction and directionally subcloned into the pLenti6/V5 lentiviral vector as recommended by the supplier (Invitrogen, Carlsbad, CA). They were packaged into infectious viral particles in 293FT cells and transduced into recipient cells followed by selection in blasticidin. A549-shRNA cells were derived from the A549 non-small cell lung cancer cell line by transduction with a pTRIPZ lentiviral vector (Open Biosystems, Huntsville, AL) encoding a DOX-regulatable bi-cistronic tran- script for RFP and a shRNA directed against human Myc. Control cells were transduced with the same vector encoding a scrambled shRNA. Stably transfected cells were selected in puromycin and those expressing the highest levels of RFP following overnight exposure to DOX (1 mg/ml) were further selected by fluorescence- activated cell sorting. The cells were then expanded in DOX-free medium. All cell lines were propagated in Dulbecco’s-modified minimal essential medium (D-MEM) containing 10% fetal bovine serum, glutamine, and penicillin plus streptomycin as previously described [67,68]. The working concentration of DOX was 5 mg/ ml and the media was replaced every 48 hr. All retroviral and lentiviral work was approved by the University of Pittsburgh Institutional Biosafety Committee and was performed under BSL2+ conditions. Fluorescence microscopy Low power (206) fluorescent images were obtained using a Zeiss Axiovert fluorescent microscope by direct observation of cells grown on tissue culture dishes and maintained at 37uC and 5% CO2. High power (606) images of cells grown to 50% confluency Myc Influences Mitochondrial Dynamics on glass bottom 6-well tissue culture plates were obtained on a Zeiss LSM710 confocal microscope at 0.5 mm intervals. Observa- tion of mitochondria was possible following staining with MitoTrackerH Deep Red FM as described below. The confocal stacks of the mitochondria were uploaded into Imaris (Bitplane Scientific Software) and a 3D reconstruction of the cells was obtained. A surface was then placed over the MitoTracker positive stain using the Imaris software. 3D movies of the reconstructed mitochondria were then generated. Electron microscopy Cells were grown to confluency in 12-well tissue culture dishes and fixed with 2.5% glutaraldehyde. Samples were prepared as previously described [69] and subsequently were photographed using a JEM 1,210 transmission electron microscope (JEOL, Peabody, MA) equipped with a CCD camera (Advanced Microscopy Techniques Corp., Danvers, MA) at 80 kV. Quantitation of mitochondrial mass and membrane potential Mitochondrial mass was measured by staining live cells with NAO and MitoTrackerH Green (Invitrogen, Carlsbad, CA). Cells were grown to 50–75% confluency in a 6-well dish. The dyes were diluted in RPMI media (final concentrations: MitoTrackerH Green = 0.5 mM, NAO = 20 nM) and exposed to the cells for 30 minutes at 37uC. Cells were washed with PBS, collected by scraping and analyzed using a FACStar flow cytometer (Becton- Dickinson Biosciences, San Jose, CA). To measure mitochondrial membrane polarization, 16106 cells were suspended in 1 ml PBS and incubated with JC-1 dye (Invitrogen) at 0.2 mM for 30 minutes. Cells were pelleted by centrifugation and re-suspended in 500 mL PBS. The cells were analyzed by flow cytometry measuring both green and red fluorescence. Relative degrees of mitochondrial polarization were quantified by measuring the ratio of red-shifted JC-1 aggregates, which are favored under conditions of high membrane potential, and green-shifted monomers, which tend to predominate under conditions of low membrane potential [41]. Additionally, cells stained with MitoTrackerH Deep Red FM (final concentration 0.5 mM) were used to assess both mass and membrane potential. PEG-Fusion Assay 56105 MycER cells expressing mito-GFP were co-plated with the same number of MycER cells expressing mito-DsRed onto glass coverslips in 12 well plates. The following day, the coverslips were incubated with DMEM+cycloheximide (CHX, 33 mg/ml, Sigma) for 30 min to inhibit de novo synthesis of fluorescent proteins. Next, coverslips were incubated in 0.5 ml PEG 1500 (Sigma) for 1 min at room temperature, washed with DMEM+CHX three times, and subsequently incubated in the same medium at 37uC and 5% CO2. One or two hours later, coverslips were fixed for 30 min with ice-cold 3.7% formaldehyde in PBS, stained with DAPI and mounted onto glass microscope slides. A minimum of 20 heterokaryons were observed using confocal fluorescence microscopy from which the percentage of co-localized fluorescence was calculated using CoLocalizer Pro software (CoLocaliztion Research Software; [70,71]) and ex- pressed as the percentage of mitochondrial fusion. Measurements of cellular OXPHOS and glycolysis All measurements were performed with a Seahorse Bioscience XF24 Extracellular Flux Analyzer (Billirica, MA). Cells were plated at 20,000 cells/well onto Seahorse 24 well plates 12– PLoS ONE | www.plosone.org 10 May 2012 | Volume 7 | Issue 5 | e37699