Fitc labeled annexin 5 apoptosis detection kit

The fluorescein isothiocyanate (FITC)-labeled Annexin V Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to quantify apoptotic cells. ACHN and A498 cells were collected after treatment with various concentrations of MET and JS-K for 24 h, then washed with PBS, and resuspended in binding buffer. Subsequently, cells were stained with FITC-labeled annexin V and propidium iodide for 15 min at room temperature in dark. The flow cytometry BD FACSDiva 6.1 software was used to analyze the stained cells within 1 h. The excitation wavelength of the fluorophore was 488nm, the emission wavelength of the fluorophore was PI: 564-606nm, FITC: 515-545nm.

The FITC-labeled Annexin V Apoptosis Detection Kit was obtained from BD Biosciences (Canada). The ROS assay kit (DCFH-DA) was purchased from Meilun (China). The cell culture medium was obtained from HyClone (Utah, USA), and cell-cultured grade fetal bovine serum (FBS), penicillin/streptomycin, and trypsin were purchased from Gibco (Thornton, Australia). The antibodies of p-Erk1/2, Erk1/2, p-Akt, Akt, cleaved-caspase 3, caspase 3, cleaved-caspase 9, caspase 9, β-actin, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-nicotinic acetylcholine receptor α7 antibody was purchased from Abcam (ab216485, Abcam). The drugs were obtained from the following sources: nicotine, methyllycaconitine citrate (MLA), dihydro-β-erythroidine hydrobromide (DHβE), and PD0325901 from MedChemExpress (MCE, USA) and H₂O₂ and N-acetylcysteine (NAC) from Sigma–Aldrich (St. Louis, MO, USA).

Extracellular ATP, PD0325901, rapamycin, and chloroquine (CQ) were purchased from MedChemExpress (MCE, San Rafael, CA, United States), and L-glutamate was obtained from Sigma–Aldrich (St. Louis, MO, United States). MRS2500 tetraammonium salt was purchased from Tocris (Bio-techne, Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, trypsin and phosphate-buffered saline (PBS) were purchased from Gibco (Thornton, NSW, Australia). The Cell Counting kit (CCK-8) was purchased from Dojindo Laboratory (Kumamoto, Japan). The JC-1 assay kit (5,50,6,60-tetrachloro-1,10,3,30-tetraethyl-imidacarbocyanine iodide) and RIPA lysis buffer were obtained from Beyotime Biotech (China). The ROS assay kit (DCFH-DA) was purchased from Meilun (China). The FITC-labeled Annexin V Apoptosis Detection kit was purchased from BD Biosciences (Canada). The antibodies of p-Erk1/2, Erk1/2, p-Akt, Akt, LC3, SQSTM1/p62, GAPDH, FITC and horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse antibody were obtained from Cell Signaling Technology (Danvers, MA, United States). The second antibody conjugated anti-mouse IgG and conjugated anti-rabbit IgG were purchased from Thermo. Anti-P2Y1 receptor antibody was purchased from Alomone Labs (Jerusalem BioPark, Israel).