*3.2. Increased Mitochondrial ROS Levels in J82 Cells Treated with Scabertopin*

We detected the cells treated with scabertopin for 24 and 48 h using a DCF fluorescent probe and found that scabertopin treatment could significantly increase the content of intracellular ROS (Figure 2A). To determine the mechanism of ROS production caused by scabertopin, we used a JC-1 assay to observe the changes of ΔΨ in the J82 cells treated with scabertopin. The results showed that ΔΨ decreased (red fluorescence decreased while green fluorescence increased) after scabertopin treatment (Figure 2C,D). In conclusion, the ΔΨ of the scabertopin treatment group decreased significantly compared to the control group.

The ROS in mitochondria are usually in the form of hydrogen peroxide (H2O2) and superoxide anions (O2 −). However, H2O2 is hardly able to escape through the mitochondrial membrane and be detected in the cytoplasm, and, thus, superoxide anion produced in the mitochondria and transported to the cytoplasm has become a key signaling factor for mitochondrial ROS [31]. Therefore, we used a DHE fluorescent probe to detect the content of superoxide anion in the cells treated with scabertopin. The results showed that the level of superoxide anion in the scabertopin treatment group was significantly higher than that in the control group (Figure 2B).

**Figure 2.** Increased mitochondrial ROS and reduced mitochondrial membrane potential in J82 cells treated with scabertopin. (**A**) Scabertopin induces ROS production in a dose- and time-dependent manner. (**B**) The DHE probe assay shows that superoxide anion is positively correlated with the scabertopin dose. (**C**) The JC-1 assay shows that, after 24 h treatment, scabertopin decreases the mitochondrial membrane potential in J82 cells in a dose-dependent manner. (**C**) The JC-1 assay shows that NAC treatment can restore the mitochondrial membrane potential reduced by 24 h treatment of scabertopin to some extent. (**D**) The DHE probe assay shows that superoxide anion is positively correlated with the scabertopin dose. (**E**) A 24 h treatment with 10 μM of scabertopin is efficient in depleting GSH through a dose-dependent mechanism in J82 cells, which can be reversed with NAC treatment. A total of 5 μM of NAC was used in the experiments. Data represent the mean ± standard error of mean (s.e.m.) of the three independent experiments. \*\* *p* < 0.01, \*\*\* *p* < 0.001, ns: no significance vs. 0 μM scabertopin-treated group. NAC: *N*-acetylcysteine; SA: scabertopin.

In addition, GSH is an important antioxidant in cells, and we also detected the changes in intracellular GSH after scabertopin treatment (Figure 2E). The results showed that scabertopin treatment could deplete GSH, which is one of the reasons for the accumulation of intracellular ROS, and the efficacy of scabertopin depleting GSH could be blocked by NAC (Figure 2E).
