Annexin 5 alexa fluor 568 conjugate

Apoptotic cells were detected by staining with annexin V-Alexa Fluor 568 conjugate (A13202, Thermo Fisher Scientific).^{19 (link)} Briefly, parental or stable ETNK2 KO GC cell lines (1 × 10⁵ cells/ml) were mixed with 10 µl of annexin V conjugate and incubated for 15 min. Cells irradiated with ultraviolet light for 120 min served as a positive control. The cells were visualised by phase contrast and fluorescence microscopy using a BZ9000 microscope (Keyence, Osaka, Japan). To quantify the percentage of cell apoptosis, the total number of cells and annexin V-positive cells in eight randomly selected fields were counted. To measure mitochondrial transmembrane potential and total caspase activity, 5.0 × 10⁴ cells/condition were collected and analysed using a Muse MitoPotential Kit or a Muse MultiCaspase Kit (Merck Millipore, Billerica, MA, USA), respectively, according to the manufacturer’s protocols.^{20 (link),21 (link)}

Cell proliferation was analyzed using the Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Apoptosis was measured using an annexin V-Alexa Fluor 568 conjugate (A13202, Thermo Fisher Scientific). Total caspase activity was measured using a Muse Multi-caspase kit (MCH100109, Merck Millipore, Billerica, MA, USA). A Caspase Colorimetric Assay Kit (BioVision, Milpitas, CA, USA) was used to measure the activity of caspase-3, 8, 9, and − 12 individually. The Muse MitoPotential kit (Merck Millipore) was used to assess the mitochondrial membrane potential. Cell cycle progression was analyzed using a Muse Cell Cycle Kit (Merck Millipore). We also analyzed the cell cycle using a Cell Cycle Assay Cell-Clock Kit (Biocolor, Carrickfergus, UK).