*2.4. Mitochondrial Reactive Oxygen Species (ROS) Are Increased in CPT1A-OE Cells*

Since GSH homeostasis was significantly changed in OE cells, we reasoned that mitochondrial ROS production could be altered in response to increased CPT1A expression. Thus, we next studied the amount of ROS produced in the mitochondria of the cell models. Figure 4A shows the stacked traces of the Electron Paramagnetic Resonance (EPR) assay, highlighting the increased amplitude of the signal in the OE cells compared to all the other samples. Quantification of the signals is shown in Figure 4B, with the OE cells having a 4-fold increase in mitochondrial ROS production compared to KD cells. Examination of the RNAseq data indicated that SOD2, the mitochondrial superoxide dismutase, was significantly decreased in OE cells compared to KD cells (1.4-fold less *p* < 0.0007, Figure 4C). The cytosolic counterpart SOD1 was not significantly changed between OE and KD cells, while the extracellular SOD3 mRNA was significantly increased in the KD compared to the OE cells (*p* = 0.01). Thus, less SOD2 expression in the OE cells likely accounted for the increase in mitochondrial ROS in these cells. Since the OE cells did not show signs of apoptosis induced by excess ROS we next challenged the cells with long chain lipids (oleic and palmitate mixture at 25 μM each) in the presence or absence of androgens, using regular fetal bovine serum (FBS) or charcoal stripped serum (CSS) devoid of steroid hormones. This fatty acid mixture was used because it represents the most common fatty acids circulating in blood and they are substrates for CPT1A activity (Figure 1D). Figure 4D,E and Figure S4, show that SOD2 did not change in response to the lipid stimulation (Figure 4D), but it decreased in the KD cells compared to the OE cells with androgen withdraw conditions (Figure 4E). Thus, in the presence of lipids and androgen deprivation conditions, CPT1A OE cells are likely to cope with the excess mitochondrial ROS from fat oxidation (Figure 4F).

**Figure 4.** Mitochondrial reactive oxygen species (ROS) are increased in CPT1A-OE cells. (**A**) Electron paramagnetic resonance spectroscopy (EPR) traces of the four cell lines tested, including the mitochondrial probe by itself. Amplitude and linewidth provide information of the amount of ROS. The concentration was acquired by Spin Fit followed by SpinCount module (Bruker). (**B**) Quantification of the EPR traces normalized to protein content. \*\*\* *p* < 0.001. (**C**) Gene expression analysis of OE versus KD cells for SOD1 ns), SOD2 (*p* = 6.9 <sup>×</sup> 10<sup>−</sup>4), and SOD3 (*p* = 0.015). Only SOD2 is a mitochondrial enzyme. (**D**,**E**) Western blots of SOD2 expression after incubation with fatty acids (Oleic and palmitate mixture; 25 μM each) in FBS (**D**) or charcoal stripped serum (CSS) (**E**) for 48 h. (**F**) Schematic of the role of SOD2 in metabolizing superoxide and the role of glutathione in eliminating H2O2. GPX = Glutathione peroxide. GSSG = oxidized glutathione.
