The distribution of J-aggregates (FL2-H channel, Red Flu) in N20 cells decreased compared to C2 cells whereas that of the JC-1 mononer (FL1-H channel, Green selleck compound Flu) increased, indicating decreased ����m in N20 cells (Fig. S4d and S4e). The population of cells that resided at the top right (UR) and the bottom right quadrant (LR) was compared in Quadrant statistics. Cell population at the LR increased in N20 (2.34%) compared to C2 cells (0.46%). These results indicate disruption of the mitochondrial membrane potential by down-regulation of NEFH. A metabolic switch from mitochondria-based, oxygen-dependent ATP production (oxidative phosphorylation) to aerobic glycolysis leads to oxygen- and mitochondria-independent ATP generation, which is a hallmark of aggressive cancer growth [38].

To determine whether the reduction in ����m associated with NEFH loss reflects a reduction in respiration, the cellular O2 level was measured. As compared to C2 cells, we observed a decrease of cellular O2 consumption (-��O2) in N12 and N20 cells with a slow slope in O2 reduction as a function of time (Fig. 4a). Measurement of intracellular ATP content revealed reduced ATP synthesis in N12 and N20 cells (40% decrease) (Fig. 4b). Energy production by O2 consumption is required for cell survival, but dysfunctional mitochondria generate reactive oxygen species (ROS), resulting in cell dysfunction or death. The level of ROS was measured using a fluorescent dye, DCDHF-DA. The baseline ROS level in both N12 and N20 cells was about 65% of that in C2 cells (Fig. 4c).

To investigate cell response to oxidative stress, cells were exposed to H2O2 in serum-free conditions for 2 hrs, and the ROS level was immediately measured. The distribution of ROS in C2 cells was shifted toward increased ROS by H2O2 treatment, whereas little change in N12 and N20 cells was observed. In separate experiments, cells were exposed to different concentrations of H2O2, and the ROS level was measured after 24 hrs of recovery. Consistent results were observed in N20 cells that exhibited reduced level of ROS compared to control (Fig. 4d). These results highlight that the cellular capacity to remove ROS increases when NEFH is lost, possibly resulting in increased resistance to oxidative stress (See Figure S4a�CS4c). Figure 4 Increase of mitochondrial dysfunction and glycolysis by decreased NEFH.

The metabolic shift results in increased lactate production via cycling through the pentose phosphate pathway, and plays an important role in Brefeldin_A malignant transformation of cancer cells. We thus collected growth medium from cell culture for examination of lactate and glucose levels. An increase of over 40% in lactate production and glucose consumption was observed in N12 and N20 cells (Fig. 4e). Taken together, loss of NEFH expression resulted in decreased ROS production and increased aerobic glycolysis.