Annexin 5 fluorescein isothiocyanate

A549 cells (5×10⁵/well) were plated in 6-well plates and cultured overnight. Subsequently, cells were treated with different concentrations of MA (0, 9, 12, 15, 18 and 21 µg/ml) for 24 h and were harvested by 0.25% trypsin. The corresponding culture medium was used as the empty control. For Annexin V/propidium iodide (PI) apoptosis analysis, the cells were resuspended in 500 µl of binding buffer and adjusted to 1×10⁶/ml. Staining solution containing 5 µl Annexin V/fluorescein isothiocyanate and 5 µl PI (Nanjing KeyGen Biotech. Co., Ltd.) was added to the cells and then incubated at 2–8°C for 15 min in the dark. Following this, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). CellQuest version 5.1 software (BD Biosciences, San Jose, CA, USA) was used to analyze the data. Each experiment was performed in triplicate.

GA (>98% purity, Sigma-Aldrich, St Louis, MO, USA) was soluble in dimethyl sulfoxide and conserved at −20°C. The preservation solution was diluted to different concentrations in use, in which the dimethyl sulfoxide concentration was <0.1%. Cisplatin was purchased from Stockhausen Pharmaceutical Co., Ltd (Lianyungang, Jiangsu, People’s Republic of China). Roswell Park Memorial Institute medium 1640 was obtained from HyClone (Logan, UT, USA). Cell Counting Kit-8 (CCK-8) was acquired from Dojindo (Rock-ville, MD, USA). Propidium iodide (PI), RNase A, and annexin V-fluorescein isothiocyanate was purchased from Keygen Biotechnology (Nanjing, Jiangsu, People’s Republic of China) and dissolved in phosphate buffered saline (PBS). The primary antibodies against Bcl-2, Bax, anticaspase-9, anticaspase-3, multidrug resistance-associated protein 2 (MRP2), lung resistance protein (LRP), and β-tubulin were acquired from Cell Signaling Technology (Danvers, MA, USA). And goat antimouse or antirabbit IgG-HRP secondary antibody was acquired from Beyotime Biotech (Nanjing, Jiangsu, People’s Republic of China).

SACC-83 cells (2×10⁵ cells/well) were seeded in 6-well plates and treated with simvastatin (0, 10, 30 and 50 µM) for 48 h. Cells were then harvested and washed twice with PBS. Cells were subsequently resuspended with 500 µl binding buffer (Nanjing KeyGen Biotech Co., Ltd.), and stained with 5 µl PI (Nanjing KeyGen Biotech Co., Ltd.) and 5 µl Annexin V-fluorescein isothiocyanate (Nanjing KeyGen Biotech Co., Ltd.) and incubated at room temperature for 10 min in the dark. In each group, 1×10⁵ cells were analyzed by flow cytometry using BD FACSDiva version 8.0.1 software (Facs Canto II, BD Biosciences).

After 4 h of transfection, cells were plated in 6-well plates (1×10⁶/well) in DMEM with 10% FBS for 48 h at 37°C and subsequently washed with PBS. Cells were resuspended in binding buffer (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) and stained with annexin-V fluorescein isothiocyanate (5 µl) and propidium iodide (5 µl; KeyGen Biotech Co., Ltd.) for 15 min in the dark at room temperature. Samples were analysed using a Beckman Coulter flow cytometer (Beckman Coulter, Inc., Fullerton, CA, USA) and FlowJo software (version 7.6.1; FlowJo LLC, Ashland, OR, USA).

Cell survival and apoptosis were assessed by flow cytometry with Annexin V-fluorescein isothiocyanate and propidium iodide (KeyGEN BioTECH) staining. Analysis of phosphatidylserine on the outer leaflet of apoptotic cell membranes was performed using Annexin V-fluorescein isothiocyanate and propidium iodide to identify apoptotic and necrotic cells, respectively. Briefly, the cells were collected with ethylenediaminetetraacetic acid-free trypsin and washed with PBS three times. Then, the cells were resuspended in 500 µl of HEPES buffer, mixed with 5 µl of Annexin V-fluorescein isothiocyanate and 5 µl of propidium iodide, and incubated at room temperature for 5 min in the dark. The cells were analyzed using a flow cytometer (BD Biosciences).