Annexin 5 fitc pi

Annexin V-FITC/PI (Keygen, Nanjing, China) was used following the manufacturer’s instructions. CNE1 and CNE2 cells were seeded in 6-well plates. For cells in which ACE was overexpressed or knocked down, radiotherapy was performed 48 h after the completion of transfection. For drug-treated cells, radiotherapy was performed 12 h after treatment with enalaprilat or DMSO. The dose of radiotherapy was 4 Gy, and cells were collected 48 h after radiotherapy. A CytoFLEX flow cytometer (Beckman, Brea, CA, USA) was used to detect apoptotic cells.

Icaritin was purchased from Sigma-Aldrich (MO, United States). Fetal bovine serum (FBS) was purchased from Gibco (Waltham, MA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), crystal violet, a cell-based ROS assay kit, N-acetylcysteine (NAC), and a senescence-associated β-galactosidase (SA-β-Gal) kit were obtained from Beyotime Biotechnology (Beijing, China). Annexin V-FITC/PI and propidium iodide (PI) were purchased from KeyGEN BioTECH (Nanjing, China). The primary antibody against γ-H2AX was obtained from Cell Signaling Technology (MA, United States). Primary antibodies against ki67, GAPDH, PARP1, cyclin D1, CDK2, CDK4, Bax, Bcl-2, P53, P21, p-EGFR, EGFR, p-AKT, AKT, and HIF1α were obtained from Proteintech (Rockville, MD, United States). Antibody against horseradish peroxidase (HRP)-conjugated secondary antibodies (goat-anti-mouse or goat-anti-rabbit) were obtained from ZSGB-BIO (Beijing, China).

Apoptosis was assessed using the Fluorometric TUNEL System from Promega, as per the manufacturer’s instructions. Briefly, the indicated cells were treated and stained using the apoptosis detection kit Annexin V-FITC/PI from KeyGen (China) according to the manufacturer’s instructions. Finally, apoptosis was detected by flow cytometry. Analysis of more than 1 × 10⁶ cells was performed per measurement.

After treatment, cells were stained with combined annexin V-FITC/PI (KeyGEN, Nanjing, China) according to the manufacturer’s instructions. Briefly, resuspend HeLa cells were mixed with 500 μl binding buffer, followed by addition of 5 μl annexin V-FITC and 5 μl propidium iodide, and then incubated for 10 min, and the apoptotic cell population was determined by flow cytometry (Becton Dickinson, Franklin, New Jersey, USA).

Annexin V-FITC / PI were used to stain cells (KeyGEN Biotech, Nanjing, China) according to the manufacturer's protocol. FACScan flow cytometer has been used to detect the rate of apoptosis (Becton Dickinson, Franklin Lakes, NJ, USA).

Warangalone was purchased from Shanghai Yuanye Bio-Technology (Shanghai, China); molecular formula, C₂₅H₂₄O₅; molecular weight, 404.462; analysis of standard substance by high-performance liquid chromatography (HPLC) ≥ 98%. Dulbecco's modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin-streptomycin, trypsin, and TRIzol were purchased from Gibco-Invitrogen (Carlsbad, CA, USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), 4',6-diamidino-2-phenylindole (DAPI), monodansylcadaverine (MDC), dimethyl sulfoxide (DMSO), 3-methyladenine (3-MA) and chloroquine (CQ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against GAPDH, LC3, BAX, BCL-2 and PARP were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against VDAC1 and PINK1 were purchased from ABclonal Technology (Boston, MA, USA). Antibodies against P62 and Parkin were purchased from Proteintech (Chicago, IL, USA). Hoechst 33342 solution was obtained from Beyotime Biotechnology (Shanghai, China). Cell cycle detection, reactive oxygen species (ROS) analysis, Annexin V-FITC/PI, and mitochondrial membrane potential detection kits were purchased from KeyGEN BioTECH (Nanjing, Jiangsu, China). MitoTracker Red CMXRos and LysoTracker™ Green DND-26 were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

For the annexin V-FITC/PI assay, MCF-7/paclitaxel cells were collected after a 72-h treatment and stained with FITC-labeled annexin V and PI according to the manufacturer's instructions (KeyGEN Biotech Inc., China). The fluorescence levels were analyzed by flow cytometry (Beckman Coulter, USA).