Annexin 5 and propidium iodide kit

Annexin V and propidium iodide kit (BD Biosciences, USA) was used to determine cell apoptosis by flow cytometry according to the manufacturer's instructions. The H446 cells treated with alone or both of anlotinib and 5-FU at IC50 levels for 24 h were harvested, washed with PBS, and then stained with annexin V and PI for 15 min at room temperature (RT) in the dark. The samples were immediately detected by a flow cytometry (CytoFLEX, Beckmen).

The analysis of cell cycle was performed as follows. At indicated times after transient transfection of PANC-1 cells with Mina53 siRNA, floating cells were collected by pipetting and adherent cells by trypsinization. Both cells were combined and fixed with 2 % paraformaldehyde followed by permeabilization with 70 % ethanol. Cells were treated with RNase A (0.25 mg/ml) at 37 °C for 30 min and stained with propidium iodine (50 μg/ml). Cellular DNA content was analyzed using flow cytometer (BD Biosciences, Franklin Lakes, USA). Cell cycle profiles were determined with CELLQuest™ software (BD Biosciences). For apoptosis analysis, cells were simultaneously stained with Annexin V and propidium iodide kit (BD Biosciences) according to the manufacturer’s instructions. Stained cells were analyzed by flow cytometry to quantify the number of apoptotic cells.