## 3 Results
### Plasma-Exposed Mushroom-Derived Extracts Induced ROS Generation for Apoptosis  
Several natural compounds from plant extracts can increase intracellular ROS levels as primary mechanisms to stimulate cancer cell death [28; 29]. Consequently, phytocomponents released from plasma-treated mushrooms might increase intracellular ROS levels as a precursor to cell death. We measured ROS levels in MCF7 cancer and MCF10A normal cells using H2 DCFDA, which emits green fluorescence in the presence of ROS upon oxidation. Figure 7A shows that intracellular ROS levels significantly increased in MCF7 cells incubated with 50 and 200 \(\mathrm{\SIUnitSymbolMicro g}\) mL\({}^{-1}\) extracts derived from plasma-treated mushrooms using flow cytometry. The untreated controls also induced ROS induction at 200 \(\mathrm{\SIUnitSymbolMicro g}\) mL\({}^{-1}\) of extract, which was consistent with the cell cycle data (Figure 7B). Levels of ROS were slightly affected in MCF10A cells subjected to similar treatments (Figure 7C,D). Microscopy analysis further confirmed the induction of oxidative stress in MCF7 cells incubated with extracts visualized as increased green fluorescence (Figure 7E). Figure 6: Cell cycle arrest is associated with cancer cell death. (**A–C**) Flow cytometry analysis of MCF7 breast cancer cells treated with extracts prepared from 0 s (untreated), 30 and 60 s plasma-treated mushrooms at 50 and 200 \(\mathrm{\SIUnitSymbolMicro g}\) mL\({}^{-1}\) using PI/RNase A, after 24 h post-incubation. Representative graph shows percentage of cell population in G2/M and S phase. *\(p<0.05\), and ***\(p<0.001\). These findings show that ROS induction can influence cancer cell apoptosis, which is regulated by several signaling pathways and related molecules. Therefore, to verify the ability of the mushroom extracts to induce breast cancer cell apoptosis, we measured the expression of apoptosis-related gene markers in MCF7 cells under all three treatment groups (0, 30, and 60 s) at a 200 \(\upmu\)g mL\({}^{-1}\) extract concentration. The expression of proapoptotic _BAX_, _p53_, _Puma_, _CytC_, _Casp3_, _Casp7_, and _Casp8_ genes were upregulated in the 30 and 60 s groups, whereas the antiapoptotic _Bcl-xL_ gene expression was not significantly altered as compared with the controls. Expression of the _Bcl2_ gene was increased in the 30 and 60 s groups. This was notable because Bcl2 belongs to the Bcl-2 family of proteins that regulate apoptotic cell death, either by inhibiting (antiapoptotic _Bcl-xL_, _Mcl-1_, _Bcl-w_, and A-1) or inducing (proapoptotic _Bax_, _Bik_, _Bak_, and _Bok_) apoptosis [30, 31]. These effects were more prominent in MCF7 breast cancer cells incubated with 30 s extracts. We also found that _H2 AX_ expression increased, indicating double-strand breaks in the DNA of MCF7 cells incubated with 30 and 60 s extracts (Figure 8A-J). These results were further confirmed by immunofluorescence staining, which revealed \(\gamma\)H2 AX foci formation in MCF7 cells treated with 30 and 60 s extracts (200 \(\upmu\)g mL\({}^{-1}\)) (Figure 9). To further confirm whether this induction of \(\gamma\)H2 AX was due to generally increased levels of H2 AX, we measured total levels of \(\gamma\)H2 AX. The levels of \(\gamma\)H2 AX did not considerably change in the cells incubated with 30 and 60 s extracts (200 \(\upmu\)g mL\({}^{-1}\)) as compared with controls (Supplementary Figure S3). Figure 7: Plasma-treated mushroom-derived extracts induced intracellular oxidative stress. (**A–D**) Flow cytometric detection of intracellular ROS in MCF7 breast cancer and MCF10A normal cells treated with extracts prepared from 0 s (untreated), 30 and 60 s plasma-treated (0 s) mushrooms, after 24 h post-incubation at 50 and 200 \(\upmu\)g mL\({}^{-1}\) using fluorescent H2 DCFDA dye, respectively; (**E**) Microscopic images of ROS detection in MCF7 cells treated in similar conditions as above. Untreated sample (no extract) was used as control for normalization. * \(p<0.05\), ** \(p<0.01\), and *** \(p<0.001\).