## 2 Results
### DSF/Cu Promotes NPC Cell Apoptosis Via ROS/MAPK Pathways  
RNA-seq analyses were performed to evaluate the transcriptome changes in 5-8F cells in response to 4 h treatment with DSF/Cu (1 uM/1 uM). More than 58,000 genes were analyzed; 1179 genes were up-regulated while 399 genes were down-regulated (Figure 4A and Figure S3A). (Kyoto encyclopedia of genes and genomes) KEGG analysis further showed that transcriptional misregulation in cancer, the p53 signaling pathway, the MAPK signaling pathway, and the ferroptosis pathway were involved in DSF/Cu-induced apoptosis or death (Figure 4B). Figure 3: Killing effect of DSF/Cu on tumor cells is ALDH-independent. (**A**) The mRNA expressions of ALDH1A1 and ALDH were detected by RT-qPCR; (**B**) ALDH1A1 and ALDH2 protein expressions were detected by Western blot in NPC cell lines and NP69 cells. (**C**) ALDH1A1 and ALDH2 proteins were detected by Western blot in NP69 cells. NP69 cells were exposed to DSF/Cu (1 uM/1 uM) for 6 h. Data are shown as means \(\pm\) SD, \(n=3\). (**D,E**) RT-qPCR and Western blotting were used to measure the expression of ALDH2 in 5-8F and CNE2 cells after transfection with siALDH2 or siNC for 48 h. Data are shown as means \(\pm\) SD. ***\(p<0.001\), \(n=3\). (**F**) 5-8F and CNE2 cells were exposed to DSF/Cu (1 uM/1 uM) after transfection with siRNA against ALDH2 (siALDH2-1) or control siRNA (NC) for 48 h. After the indicated time points, inhibition effects were determined by MTT assay. (**G**) Cells were exposed to different drugs for 24 h. The inhibition effects were determined by MTT assay. A549/CDDP (cisplatin) was used as ALDH1A1 positive cell control. Data are shown as means \(\pm\) SD. ***\(p<0.001\), \(n=3\). Figure 4: DSF/Cu induces antitumor activity through a joint action of multiple pathways. (**A**) Gene expression heatmap showing differentially expressed genes in 5-8F after being exposed to DSF/Cu (1 uM/1 uM) for 4 h. (**B**) Part of the unregulated significantly differentially expressed gene pathway bubble map; the processing conditions were the same as in (**A**). (**C,D**) 5-8F cells were cultured with DSF/Cu (1 uM/1 uM), and the protein expressions were detected by Western blot; the JNK inhibitor SP600125 (10 uM) and p38 inhibitor SB203580 (10 uM) were pretreated 12 h before DSF/Cu was added. Data are shown as means \(\pm\) SD. * \(p\) \(<\) 0.05, ** \(p\) \(<\) 0.01, *** \(p\) \(<\) 0.001, \(n\) = 3. (**E**) Cellular ROS production in 5-8F treated with DSF/Cu (1 \(\upmu\)M/1 \(\upmu\)M) and the ROS inhibitor NAC (10 mM) for 5 h was assessed by flow cytometry using DCFH-DA. NAC was pretreated for 12 h. Data are shown as means \(\pm\) SD. ** \(p\) \(<\) 0.01, *** \(p\) \(<\) 0.001, \(n\) = 3. (**F**) Representative images from the High Content screening (HCS) assay of 5-8F cells treated with DSF/Cu, DSF/Cu plus the ROS inhibitor NAC (10 mM) for 5 h. NAC was pretreated for 12 h before DSF/Cu was added. Images with magnification at 10 \(\times\) are shown here, scale bar: 100 \(\upmu\)M. Quantification of cellular ROS levels from the HCS assay are shown as means \(\pm\) SD. * \(p\) \(<\) 0.05, *** \(p\) \(<\) 0.001 vs. control group. ## \(p\) \(<\) 0.01, ### \(p\) \(<\) 0.001 vs. DSF/Cu group (the second group), \(n\) = 3. The apoptosis-related MAPK pathway was verified first. The mRNA levels of eight MAPK-related genes (CACNA1G, DUSP1, NR4A1, HSPA2, GADD45G, HSPA6, HSPA1B, HSPA1A) were significantly increased after DSF/Cu (1 \(\upmu\)M/1 \(\upmu\)M) treatment in 5-8F cells (Figure S3B). Similar results were also observed in the mRNA expression of four key genes in the MAPK pathway (MAP2K3, MAP2K4, MAP3K1, JUN) (Figure S3C). Then, Western blot analysis was used to verify the activation of the JNK and p38 MAPK pathways. As shown in Figure 4C, phosphorylated JNK (p-JNK) and the p-JNK/JNK ratio began to increase after 10 min post-incubation in DSF/Cu-treated 5-8F cells. Similar results were observed in the expression of p-p38 and the p-p38/p38 ratio (Figure 4C).