Measurement of the Mitochondrial Membrane Potential (ΔΨm)

The mitochondrial membrane potential (ΔΨm) was assessed by the retention of Rhodamine 123, a membrane-permeable fluorescent cationic dye that is selectively taken up by mitochondria (Baracca et al., 2003). In brief, cells (1 × 10⁶) were plated in 6-well plates, cultured with or without AD0157 for 14 h and incubated with 0.1 μg/mL Rhodamine 123 in the dark for 30 min at room temperature, prior to termination of the experiment. Subsequently, cells were collected, washed with PBS and analyzed by flow cytometry, with excitation and emission wavelengths of 495 and 535 nm, respectively. The levels of Rhodamine 123 retention in mitochondria were proportional to the ΔΨm.