Apoptosis assay

Annexin V and viability dye were used to detect early apoptotic and dead cells, respectively. 24 h after the transfection cells were counted and 150,000 cells were suspended in 100 μL of 1X PBS. The viability dye (ThermoFisher #35111) was added (1:5,000) in 100 μL of 1X PBS and cells were incubated for 30 min at 4°C. Cells were then washed once with 1X PBS and suspended in 100 μL of Annexin buffer PH 7, added PE Annexin (1:200; ThermoFisher #{"type":"entrez-nucleotide","attrs":{"text":"L34960","term_id":"522203","term_text":"L34960"}}L34960), and incubated for 30 min at 4°C. After a wash with 1X PBS, cells were resuspended in 300 μL of Annexin buffer and underwent the flow analysis by using the LSR Fortessa instrument (BD Biosciences). Unstained cells were used as control.