## 2 Results
### DSF/Cu Reduces the Viability of Fibroblasts and Attenuates Fibroblast Activation  
Activated fibroblasts characterized by \(\alpha\)-smooth muscle actin (\(\alpha\)-SMA) expression are considered to be the main cellular constituents of tumor stroma. We further investigated the cytotoxic effects of  
Figure 5: DSF/Cu inhibits nasopharyngeal carcinoma cells via the ferroptosis pathway. **(A)** Western blot analysis for the expression of p53-related proteins in 5-8F treated with DSF/Cu (1 \(\mu\)M/1 \(\mu\)M); the p53 inhibitor Fifithrin-\(\alpha\) (20 \(\mu\)M) was pretreated for 12 h. Data are shown as means \(\pm\) SD. * \(p<0.05\), ** \(p<0.01\), ** \(n=3\). **(B,C)** RT-qPCR analysis of Ptgs2 and SAT1 mRNA levels in 5-8F. Data are shown as means \(\pm\) SD. * \(p<0.05\), ** \(p<0.001\) vs. control group, \(n=3\). **(D)** The expression of SAT1 was detected by Western blotting in 5-8F, after cultured with DSF/Cu (1 \(\mu\)M/1 \(\mu\)M). Data are shown as means \(\pm\) SD. *** \(p<0.001\), ** \(n=3\). **(E)** RT-qPCR analysis of ALOX15 mRNA levels in 5-8F. Data are shown as means \(\pm\) SD. ** * \(p<0.01\), ** \(p<0.001\) vs. control group, \(n=3\). **(F)** Lipid ROS production in 5-8F treated with DSF/Cu (1 \(\mu\)M/1 \(\mu\)M) for 6 h was assessed by flow cytometry using C11-BODIPY. NAC was pretreated for 12 h. Data are shown as means \(\pm\) SD. *** \(p<0.001\), \(n=3\). **(G)** The percentage of viable 5-8F cells after being treated with DSF/Cu (1 \(\mu\)M/1 \(\mu\)M) for 24 h. Inhibitors were pretreated for 12 h. DMSO solvent was used as a control. Data are shown as means \(\pm\) SD. *** \(p<0.001\) vs. DSF/Cu group (the second group), \(n=3\). DSF/Cu on \(\alpha\)-SMA-positive human skin fibroblasts (HSF). Similar to NPC and NP69 cells, DSF/Cu exhibited a highly cytotoxic effect on HSF (ALDH1A1\({}^{\text{high}}\)) in a DSF concentration-dependent manner and a time-dependent manner by MTT and LDH assays, and the level of ALDH1A1 was not obvious changed after incubation with DSF/Cu for 6 h (Figure 6A,B, Figure S5A). DSF/Cu-treated HSF showed cell shrinkage, shortened gradually from being fibrous to spherical, and eventually died (Figure 6C). In addition, both the activated fibroblast marker \(\alpha\)-SMA and fibroblast activation protein alpha (FAP-\(\alpha\)) were significantly down-regulated when treated with DSF/Cu for 4 h, suggesting that DSF/Cu treatment led to the functional inactivation of HSF (Figure 6D). Figure 6: \(Cont.\)  
Furthermore, FACS analysis showed that DSF/Cu induced apoptosis and necrosis in HSF in a time-dependent manner (Figure 6E). Surprisingly, compared with NPC cells, DSF/Cu induced HSF apoptosis more rapidly and profoundly. The apoptosis rate was about 46% in HSF after treatment with DSF/Cu for 4 h, while a similar apoptosis rate in NPC needed 10 h of treatment (Figure 6F). With 10 h of treatment, the apoptosis rate of HSF reached 73% (Figure 6E,F). Moreover, the cellular ROS levels in HSF increased after treatment with DSF/Cu for 4 h (Figure 6G). Next, we investigated the inhibitory effects of DSF/Cu on TGF-\(\beta\)1-stimulated mouse fibroblasts NIH 3T3. As shown in Figure 7A and Figure 5S8, \(\alpha\)-SMA protein was hardly detected in untreated 3T3 cells (ALDH2 high), but increased significantly after being incubated with 20 ng/mL TGF-\(\beta\)1 for 48 h. The toxicity of DSF/Cu was different between 3T3 and TGF-\(\beta\)1-stimulated 3T3 cells. The cell viability of 3T3 cells was 65%, while it was only 43% in TGF-\(\beta\)1-stimulated 3T3 cells after being incubated with DSF/Cu for 24 h (Figure 7B). Similar to HSF, DSF/Cu-treated 3T3 cells showed cell shrinkage, roundness and floating, but TGF-\(\beta\)1-stimulated 3T3 cells exhibited more obvious morphologic changes after being treated with DSF/Cu (Figure 7C). The expression of \(\alpha\)-SMA was almost lost after being treated with DSF/Cu for 12 h in TGF-\(\beta\)1-stimulated 3T3 cells (Figure 7D). These results indicated that activated fibroblasts were more sensitive to DSF/Cu than tumor cells and inactivated fibroblasts.