## 3 Results
### ASE Induced Ferroptosis Domineered in the A549 Cell Line  
ROS accumulation and lipid peroxidation are both involved in ferroptosis, ultimately leading tumor cells to undergo oxidative cell death [29]. As mentioned above, A549 cells treated with the ASE for 48 h exhibited increased numbers of 7-AAD-positive cells distinct from those Annexin-V-positive cells in the positive control, the DTX group. In addition, by evaluating morphological changes, we determined that the ASE induced a spherical morphology configuration that prevailed in A549 cells (Figure 1D,E) which was similar to morphological changes of ferroptotic cells [27]. Therefore, both intracellular ROS levels and lipid peroxidation were analyzed using DFCH-DA and C11-BODIPY assays after exposure to the ASE. As expected, the ASE (100 and 200 \(\upmu\)g/mL) triggered significant increases in ROS levels and lipid ROS activation preferentially in the A549 cell line but not in H23 cells (Figure 3A,C). Co-treatment of cells with the potent ferroptosis inhibitor, DFO, completely abrogated the ASE-induced population of ROS-positive cells and lipid peroxidation in A549 cells, but with no such effect in H23 cells (Figure 3A-C). Moreover, the results from Annexin/7-AAD double staining using a flow cytometric analysis showed that DFO could almost entirely rescue the ASE-induced cell death response in A549 cells, whereas it only partially reversed cell viability in H23 cells (Figure 3D). GPX4 was previously described as a crucial defense enzyme against lipid peroxidation, and the proper functioning of GPX4 is essential for cellular processes in preventing ferroptosis [30,31]. Hence, GPX4 expression after ASE treatment was evaluated. Results showed that the ASE decreased the GPX4 level at a significantly higher rate in ASE concentrations of 200 \(\upmu\)g/mL, principally in the A549 cell line (Figure 3E,F) with statistical significance. Conversely, there was nearly no alteration in GPX4 levels in H23 cells, as shown in Figure 3E,F. Collectively, our data showed that non-apoptotic iron-dependent ferroptosis was primarily involved in A549 cell death but not in H23 cell death. Figure 2: The ethanol extract of _Artemisia santolini_ (ASE) triggers different features of the cell death mode. Flow cytometry and Western blotting were performed to analyze the cell death mode following ASE and DTX treatment in A549 and H23 cells. (**A**) Apoptosis assay after staining with Annexin V-FITC/7-aminoactinomycin D (7-AAD). A549 and H23 cells were treated with 100 and 200 \(\upmu\)g/mL ASE or 0.5 nM DTX (the positive control) for 48 h. (**B**) Quantification of dead cells following ASE treatment. (**C**) Representative images of Western blot analysis of caspase-3 expression with the most significant alterations. \(\upbeta\)-actin was used as a loading control. (**D**) Histograms quantified Western blot results of three replicates. Data are the mean \(\pm\) SE; **\(p<\) 0.01 vs. the corresponding control. Figure 3: _Cont._  
Figure 3: Treatment with the iron chelator, desferrioxamine (DFO), prevented ferroptosis upon response to exposure to the ethanol extract of _Artemitsia santoliniifolia_ (ASE) in non-small cell lung cancer (NSCLC) cells. A549 and H23 cells were treated with DFO (100 \(\upmu\)M) 12 h prior to ASE (100 and 200 \(\upmu\)g/mL) treatment. (**A**) The ASE induced reactive oxygen species (ROS) production in A549 and H23 cells as determined by a DCFH-DA assay. (**B**) Histograms represent the effect of DFO on ASE-induced ROS production of three replicates (MFI, mean fluorescence intensity), and lipid ROS levels were measured using a C11-BODIYY assay after indicated treatment above (**C**). (**D**) Cell death populations of A549 and H23 cells following ASE treatment for 24 h in the presence or absence of the iron-chelator, DFO, as quantified by Annexin/7-AAD staining and analyzed using flow cytometry.