## 2 Results
### Membrane Permeability and Fluidity Analysis  
Previous studies have suggested that increasing membrane permeability, enhancing antibiotic uptake, can be an effective strategy for controlling persister cells. To investigate whether diosgenin affects membrane permeability in _S. aureus_, fluorescence assays were conducted using 80 uM and 160 uM diosgenin (Figure 5A). In the control group, baseline membrane permeability values remained relatively constant. Contrary to expectations, treatment with diosgenin at either concentration did not result in any significant change in membrane permeability compared to the control group. Fluorescence intensity values were statistically indistinguishable across all treatments, indicating that diosgenin does not significantly alter membrane permeability under the tested concentrations. These findings align with the observation that diosgenin does not exhibit bactericidal activity against pre-formed persister cells (Supplementary Figure S2).  
Figure 4: Effect of diosgenin on intracellular ATP levels in _Staphylococcus aureus_. _Staphylococcus aureus_ cells were cultured in 80 uM and 160 uM diosgenin media, and ATP levels were quantified by luminescence and normalized to cell density (Abs\({}_{600}\)) to calculate intracellular ATP per cell. Data are presented as means \(\pm\) the standard deviation of three independent experiments. Statistically significant differences at the 99% confidence level (\(p<0.01\)) between the treatment means and the control mean are indicated by asterisks (*******).  
Next, considering evidence that diosgenin is structurally capable of interacting with membrane lipids, its effect on membrane fluidity was assessed to determine potential impacts on membrane-associated protein activity (Figure 5B). Benzyl alcohol, a known membrane fluidizer, was used as a positive control to validate the assay's responsiveness. In cells cultured with 80 uM diosgenin, membrane fluidity decreased by 35% relative to the untreated control, and the 160 uM diosgenin treatment decreased membrane fluidity by 41%, indicating that diosgenin induces membrane rigidification in a dose-dependent manner. In contrast, benzyl alcohol increased membrane fluidity by 43%, confirming its expected fluidizing effect.  
To further explore whether diosgenin affects membrane potential, fluorescent membrane potential indicators were utilized to assess potential changes under 80 uM and 160 uM diosgenin treatment (Supplementary Figure S1). No significant differences in membrane potential were found between diosgenin-treated and control groups, suggesting that diosgenin does not substantially alter membrane polarization at the tested concentrations.  
The observed reductions in membrane fluidity without concurrent increases in membrane permeability or significant changes in membrane potential suggest that diosgenin's primary mechanism of action may not involve membrane disruption but rather the modulation of membrane-associated signaling. Membrane fluidity is known to influence the activity of embedded proteins and receptors, potentially affecting stress response pathways and persister cell signaling mechanisms. Thus, the decrease in membrane fluidity induced by diosgenin may alter the functional dynamics of membrane-associated proteins, contributing to its inhibitory effect on persister cell formation.