Myc Influences Mitochondrial Dynamics Figure 2. Temporal changes in mitochondrial density and structure in response to Myc. (a) Mitochondrial Mass. MycER cells were exposed to 4-HT for up to 35 d at which point 4-HT was removed. At the indicated times, cells were stained with NAO and MitoTracker Deep Red and analyzed by flow cytometry. Values are expressed relative to control cells not exposed to 4-HT, which were arbitrarily set to a value of one. (b) Mitochondrial mass by microscopy. MycER cells stained with MitoTracker Red were observed by epifluorescence microscopy at low power. The intensity of staining is consistent with the finding from flow cytometry. (c) Mitochondrial ultrastructure. MycER cells were exposed to 4-HT for the indicated times and observed by transmission electron microscopy. Representative mitochondria from each time point are depicted. Note that the untreated cells had small, cristae-deficient mitochondrion similar to those of the myc2/2 cells. After several weeks of Myc induction the mitochondria took on a more normal and mature appearance. The size of the mitochondria began to decrease as 4-HT was removed from the media. The addition of 4-HT to cells lacking the MycER construct had no effect on any of the above-measured mitochondrial properties (not shown). (d) Mitochondrial shape. MycER cells were observed under live confocal microscopy either prior to or following the addition of 4-HT for 35 days. 4-HT was then removed and the cells were imaged again 1 day or 10 days later. Mitochondria were stained with MitoTracker Deep Red and 3-dimensional reconstructions of the cells were performed from a series of z-stacks. Shown are representative images with a space-filling overlay to emphasize the mitochondria. Note the loss of total mitochondria and the decrease in interconnectivity upon removal of 4-HT. Additional cells can be seen in a video detailing the three- dimensional structure (Video S1). doi:10.1371/journal.pone.0037699.g002 These results suggested that, despite overall higher basal levels of OXPHOS and respiratory reserve capacity of myc2/2wtMyc cells, the majority of their ATP supply is derived from glycolysis. This was confirmed by concomitant measurements of the extracellular acidification rate (ECAR) (Figure S2). These results, which measure the increased acidity of the medium as glucose is converted to lactate, showed that ECAR also correlated with Myc expression and were consistent with previous findings that constitutive ectopic Myc expression in Rat1a fibroblasts is associated with a high rate of glycolysis [43]. Relative to myc2/ 2 cells, myc2/2wtMyc cells showed approximately three-fold higher levels of ECAR (Figure S2). These results were largely reproduced in MycER cells where net ATP content was observed to increase as a function of time of Myc induction and where ATP production was completely abrogated by 2-DG (Figure 3c). The finding that myc+/+ cells and myc2/2wtMyc cells contained equivalent ATP levels seemed at first to be inconsistent with their different rates of glycolysis and OXPHOS. We therefore measured ATP half-life in each cell line and showed it to be shorter in myc2/2wtMyc cells (2.661.0 minutes) than in myc+/+ cells (3.660.8 minutes; Figure S3; p = 0.02). Taken together, these results suggest that the apparent discrepancy between ATP levels and rates of OXPHOS in myc+/+ and myc2/2wtMyc cells can likely be explained by higher rates of ATP turnover by the latter cells. Finally, we asked whether the effects of deliberately enforced Myc expression on mitochondrial structure and function could be observed in cells in which high endogenous Myc levels were a natural consequence of the transformed state. We therefore repeated several of the above studies in human A549 non-small cell lung cancer cells stably transduced with a lentiviral vector PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e37699