4 6 diamidino 2 phenylindole dihydrochloride dapi

After ROT treatment (0.5 µmol/L for 24 hours), cell apoptosis was detected with the one‐step TUNEL kit as per the manufacturer's instructions (Beyotime Biotechnology). Briefly, the cells were fixed in 4% paraformaldehyde for 15 minutes and permeabilized with 0.1% Triton X‐100 for 5 minutes. After several washes with PBS, 50 µL of TUNEL reaction mixture was added to the cells, and they were incubated at 37°C for 1 hour in the dark. Finally, the cells were examined under a fluorescent light microscope (Leica). For DAPI staining, the SH‐SY5Y cells were seeded in 35‐mm plates. After treatment, the cells were washed in cold PBS and fixed with 4% paraformaldehyde for 30 minutes. After three more washes in cold PBS, cells were incubated with a 5 μg/mL 4,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI) (Sangon Biotech) solution for 20 minutes. Finally, the cells were again washed in cold PBS three times and examined using the same fluorescent light microscope to visualize the DAPI‐stained cell nuclei.

After PRRPR-GFP and SMO-GFP were overexpressed for 48 h, HaEpi cells were washed three times with 500 μL of Dulbecco’s phosphate-buffered saline (DPBS; 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄ and 8 mM Na₂HPO₄, pH 7.4), and fixed with PBS containing 4% paraformaldehyde for 10 min in the dark at room temperature. The fixed cells were washed three times for 3 min each. The plasma membrane was stained using wheat germ agglutinin (WGA; Sigma-Aldrich, St. Louis, MO, United States; 1 μg/mL in PBS) in the dark for 4 min and then washed with PBS six times. Nuclei were stained with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI; Sangon Biotech, Shanghai, China; 1 μg/mL in PBS) in the dark at room temperature for 10 min and then washed with PBS six times. Fluorescence was detected using an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan). The negative control (GFP expression) was treated following the same method.

Rotenone was obtained from Sigma-Aldrich Co., LLC (St. Louis, MO, USA), and PA (CAS no. 4852-22-6) was purchased from Yuan Ye (Shanghai, China). Antibodies against β-actin, cleaved caspase-9, cleaved caspase-3, cleaved PARP, phospho-ERK1/2, phospho-p38, p38, phospho-JNK1/2 were purchased from Cell Signaling Technology (Beverly, MA, USA), anti-ERK2 and JNK1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Sangon (Shanghai, China). Inhibitors SB203580 (p38 MAPK) and SP600125 (JNK), were from Sigma Aldrich. Inhibitor U0126 (MEK) were from Cell Signaling Technology. The One-step TUNEL apoptosis assay kit was purchased from Beyotime (Shanghai, China). Fluorometric Intracellular ROS Kit was purchased from Sigma-Aldrich Co., LLC (St.Louis, MO, USA).