## 3 Results
### Extracts Derived from Plasma-Treated Mushrooms Induce Cancer Cell Death due to Cell Cycle Arrest  
The results of the Alamar Blue activity assay showed that the viability of cancer cells concentration dependently decreased when incubated with the extracts of plasma-exposed mushrooms. Annexin-V/PI provides a sensitive approach for determining cellular apoptosis, as well as necrotic or late apoptotic cells. Thus, we further assessed cell death in breast cancer cells, at extract concentrations of 50 (low) and 200 (high) \(\upmu\)g mL\({}^{-1}\), using flow cytometry. The Annexin-V/PI findings revealed that the plasma-treated mushroom-derived extracts for 30 and 60 s caused the maximum death of MCF7 cells as compared with the controls (Figure 5A,B). The extracts exposed to plasma for 30 and 60 s (at a concentration of 200 \(\upmu\)g mL\({}^{-1}\)) resulted in 30.25% (double-positive cells) and 20.65% cell death, respectively. The control extracts were relatively toxic toward MCF7 cancer cells (14.93% at 50 \(\upmu\)g mL\({}^{-1}\) and 12.13% at 200 \(\upmu\)g mL\({}^{-1}\)), but minimally toxic toward MCF10A cells (5.77% at 50 \(\upmu\)g mL\({}^{-1}\) and 4.36% at 200 \(\upmu\)g mL\({}^{-1}\), Figure 5C,D). The findings were similar in MDA-MB231 cancer cells but were more prominent in MCF7 cells (Supplementary Figure S2).  
Figure 4: Extracts derived from plasma-treated mushrooms are cytotoxic to breast cancer cells. (**A–C**) Results of Alamar Blue assays performed using MDA-MB231, MCF7 cancer, and MCF10A normal cells incubated for 24 h with 0, 10, 50, 100, 200, 500, and 1000 \(\upmu\)g mL\({}^{-1}\) of extracts derived from plasma-treated mushrooms for 30 s, 60 s, or left unexposed (0 s, control). \({}^{*}p<0.05\), \({}^{**}p<0.01\), and \({}^{***}p<0.001\). Untreated sample was used as a control in all tested plant extracts groups for normalization.  
Many natural products inhibit cell proliferation and induce death by inducing arrest at different phases of the cell cycle [27]. Cell cycle arrest (G1 phase) and apoptosis are different means of cellular growth arrest that lead to the failure of a cell to move into the S phase. Therefore, we analyzed MCF7 cancer cell cycles, after incubation with 50 and 200 \(\mathrm{\SIUnitSymbolMicro g}\) mL\({}^{-1}\) of extracts. Flow cytometry results revealed an arrest in the G2/M phase of MCF7 cells by PI/RNase A staining with plasma-treated mushroom-derived extracts, while S-phase was also affected at 30 s plasma-treated mushroom-derived extract at 200 \(\mathrm{\SIUnitSymbolMicro g}\) mL\({}^{-1}\). The effects were similar in MCF7 cells incubated with control extract (Figure 6A-C), indicating that the untreated mushrooms contain several phenolic compounds that affect the cell cycle, rather than promoting proliferation in cancer cells. However, this requires further investigation. The results indicated that the inhibitory effects induced by the extracts on breast cancer cell growth were due to the incidence of cell death associated with cell cycle arrest.  
Figure 5: Plasma-treated mushroom-derived extracts stimulate apoptotic cell death in breast cancer cells. (**A–D**) Cell death analysis in MCF7 breast cancer and MCF10A normal mammary epithelial cells treated with extracts prepared from 30 and 60 s plasma-treated, and untreated (0 s) mushrooms at 50 and 200 \(\mathrm{\SIUnitSymbolMicro g}\) mL\({}^{-1}\) after 24 h post-incubation using Annexin-V/PI staining.