Myc Influences Mitochondrial Dynamics Figure 4. Mitochondrial structure and function in human A549 cells. (a) Effect of Myc shRNA induction. A549 cells expressing doxycycline (DOX)-inducible Myc shRNA were exposed to DOX for 4 days after being grown to 50–75% confluency. The upper right panel shows the signal from the co-expressed RFP. The upper left panel shows the same cells grown in the absence of DOX. The lower panels represent changes in growth as a result of DOX exposure following the plating of equal numbers of cells and a subsequent four day period of growth +/24-HT. (b) Endogenous Myc protein levels. Immunoblotting for Myc protein in control, non-DOX-treated cells or those exposed to DOX for four days. b-actin was used as a loading control. (c) Mitochondrial mass. Cells were exposed to DOX for four days and stained with MitoTracker Green. (d) OXPHOS. A typical experiment as performed on the Seahorse is shown for cells grown in the presence and absence of DOX for four days. Included are control A549 transduced with a lentiviral vector expressing a scrambled shRNA sequence and which do not display any changes in OXPHOS when exposed to DOX. doi:10.1371/journal.pone.0037699.g004 Relative to myc+/+ cells, myc2/2 cells showed decreases of several respiratory complexes, most notably Complex I, whose levels were reduced approximately four-fold (Figure 5b). Less pronounced reductions were also noted in Complexes II, III, and monomeric Complex V (Vm). These results stand in contrast to the minimal change in Complex IV between these two cell lines. Mitochondria from myc2/2 cells also contained an unidentified complex (‘‘Complex X’’; Figure 5a) that was increased more than four-fold relative to the other two cell types. Preliminary analysis of Complex X by mass spectroscopy indicates that it contains some of the subunits from Complexes II–V, amongst other proteins. Some ETC complexes are also found in higher order structures, either as homodimers, or in association with other complexes. For example, Complex V occurs in both monomeric (Vm) and dimeric (Vd) forms, with the latter having been proposed to be more enzymatically efficient [49]. Similarly, all individual complexes can associate in various combinations to form more efficient super- complexes (SCs), such as those comprised of Complexes I+III+IV [50]. These higher order structures were also depleted in myc2/2 cells as evidenced by the approximately two-fold and five-fold decreases in Complex Vd and SCs, respectively (Figures 5b and 5c). Given the restoration of mitochondrial mass and OXPHOS in myc2/2wtMyc cells, it was expected that they would have similarly normal ETC complexes; rather, they showed only a partial restoration of ETC complexes. Specifically, these cells displayed levels of complexes I, II, Vm, Vd, and SCs that were intermediate to those of myc2/2 and myc+/+ cells (Figure 5a and b). Thus, the restoration of Myc by its enforced over-expression only partially corrected the ETC complex abnormalities seen in myc2/2 cells. Next, we studied the enzymatic activities of Complexes I and V using previously described in situ activity stains [48,51,52]. These studies showed that Complex I activity in myc+/+ cells resided in both the Complex I and SC bands and was decreased by nearly two-thirds in myc2/2 cells (Figure 5c). Overall, total Complex I activity was only partially restored in myc2/2wtMyc cells (Figure 5c). PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e37699