Mitoview 633 dye staining kit

The mitochondrial membrane potential (MMP) was evaluated using a MitoView^TM 633 dye staining kit obtained from Biotium, Fremont, CA, USA. According to the manufacturer’s protocol, the A549 cells (1 × 10⁶ cells/well) were plated in a 6-well plate and incubated overnight. Then, the cells were treated with various concentrations of DLEE for 24 h before being harvested. The cells were incubated with 100 nM of MitoView^TM 633 dye in incomplete DMEM for 20 min in a 5% CO₂ incubator. After incubation, the cells were washed with PBS and were analyzed using a flow cytometer (Beckman Coulter Inc., Indianapolis, IN, USA) at excitation/emission of 638/660 nm. The data were analyzed using CytExpert for DxFLEX 2.0 software.

Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), 2.5% trypsin, and penicillin-streptomycin were obtained from Gibco BRL Company (Grand Island, NY, USA). The protease inhibitor cocktail, RIPA lysis buffer, the reagent of Coomassie Plus^TM Protein Assay, and the enhanced chemiluminescence (ECL) reagent were purchased from Thermo Fisher Scientific (Rockford, IL, USA). The apoptosis assay kit was purchased from Bio-Legend (San Diego, CA, USA). The sulforhodamine B (SRB) reagent was obtained from Sigma-Aldrich, St. Louis, MO, USA. The propidium iodide (PI) dye, and primary antibody for anti-β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies for Western blot analysis (cyclin D1, cyclin E1, CDK-2, CDK-4, caspase-9, cleaved-caspase-3, Bcl-2, Bcl-xl, and HRP-conjugated anti-mouse or rabbit-IgG) were obtained from Cell Signaling Technology (Beverly, MA, USA). The primary antibody of PARP-1 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The MitoView^TM 633 dye staining kit was obtained from Biotium (Fremont, CA, USA).