Apoptosis Evaluation in A375 and A2058 Cells

The apoptotic cell population was measured by using Muse™ Annexin V and Dead Cell kit (Millipore, Burlington, MA, USA) in accordance with a previously described [51 (link)]. Initially, LEF1 stably overexpressing A375 and A2058 cells were generated with puromycin selection. Empty vector (pBABE-puro-empty vector) or LEF1 (pBABE-puro-LEF1) overexpressing A375 and A2058 cells (1 × 10⁵ cells/well) were seeded onto a 6-well cell culture plate and incubated for 48 h in the absence or presence of 100 nM of cinobufagin. After cinobufagin treatment, the cells were harvested and washed with cold PBS, and then cells were incubated in 100 μL Muse™ Annexin V and Dead Cell kit reagents (Millipore, Burlington, MA, USA) for 20 min at room temperature. Finally, the cells were subjected to Mini Flow Cytometry Muse™ Cell Analyzer (Millipore, Burlington, MA, USA) to measure apoptotic cell populations.