*2.5. Effect of JI017 Suppressed Cell Growth In Vivo*

To further confirm the efficacy of JI017 for inhibiting cell growth in animal experiments, H460 cells were subcutaneously injected into nude mice. We did not find any change in body weight in the control group and the JI017-treated group (Figure 5A). The sizes of xenograft tumors in the JI017-treated mice were smaller compared to the xenograft tumors in the control mice, and the tumor suppression rate was 79.2%. These results indicated JI107 treatment slowed the tumor growth rate (Figure 5B,C). Because ROS reacts easily with lipids, free radical formation can be confirmed through lipid peroxidation markers. The peroxidation of membrane lipids can alter physical properties, such as lipid interaction, ion

gradient, membrane fluidity, and permeability [53]. We found that JI017 treatment increased MDA concentrations in vivo (Figure 5D). Moreover, the addition of JI017 increased the levels of LC3 and cleaved caspase3 (Figure 5E). These results showed that the protein levels of Ki-67 decreased and the protein levels of cleaved caspase-3 and LC3 increased in the JI017-treated group compared to the control group (Figure 5F). Overall, the above results show that JI017 induced ROS in vivo and inhibited lung cancer cell proliferation and tumor growth.

**Figure 5.** JI017 suppressed lung cancer cell growth in mice. BALB/c nude mice were subcutaneously injected with H460 cells. (**A**) The mouse body weight and (**B**) the tumor growth rate are shown. (**C**) Representative tumor images of the control group and the JI017-treated group. (**D**) The MDA concentration was assessed by a thiobarbituric reactive substance (TBARS) assay and normalized to the protein concentration. (**E**) Whole tissue lysates were analyzed by Western blotting with anti-LC3, anti-cleaved caspase 3, and anti-GAPDH antibodies. (**F**) The IHC staining of Ki-67 and cleaved caspase-3 and LC3 was carried out. Scale bar = 200 μm for 20× and Scale bar = 100 μm for 40×. The data are expressed as the mean ± SEM in all groups (*n* = 8–11). \* *p* < 0.05 and \*\* *p* < 0.01 compared to the untreated group.
