Mitotracker red fm dye

For the measurement of mitochondrial membrane potential (ΔΨ_m), MitoTracker Red FM Dye (1 µM; Thermo Fisher Scientific, Waltham, NJ, USA) was employed. The cells were stained with DAPI and the MitoTracker in PBS for 15 min. To determine the glutathione content, the cells were stained with propidium iodide (PI; 5 µg/mL; Santa Cruz Biotechnology, Heidelberg, Germany) and ThiolTracker Violet (10 µM; Thermo Fisher Scientific, Waltham, NJ, USA) in PBS containing calcium and magnesium. The staining was executed for 30 min at 37 °C. After the staining, the cells were washed with RPMI, and the fluorescence was measured using flow cytometry at 4 h and 24 h after the treatment.

Mitochondria were stained using Invitrogen MitoTracker Red FM dye (Thermo Fisher Scientific, Inc). The cells were suspended in PMU medium, and MitoTracker Red FM was added to a final concentration of 50 nM. After incubation at room temperature for 15 min, the cells were visualized at 1000× magnification using a BX51 fluorescent microscope (Olympus). The fluorescence of GFP^S65A was observed at an excitation wavelength of 485 nm. Fluorescent images were obtained using a digital camera (DP74; Olympus) connected to the microscope. Final merging of images was done using Adobe Photoshop 2022 software.

Fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), and penicillin-streptomycin (P/S) were purchased from Welgene (Gyeongsangbuk-do, Korea). MitoTracker^™ Red FM dye was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against phosphorylated ERK 1/2 (P-ERK 1/2), ERK 1/2, phosphorylated p38 (P-p38), p38, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Manufacturing grade-certified (Korean Pharmacopoeia) SMS was obtained from Jungwoo Medicine (Chungcheonnam-do, Korea). SMS is composed of 1,250 mg GS, 1,250 mg SC, and 2,500 mg LM in 100 mL water. All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).