Annexin 5

Live-cell imaging was performed using the IncuCyte S3 Live-cell analysis system (Essen BioScience, Ann Arbor, MI, USA). Cells were seeded in a 24-wells plate at a density of 2.500–4.000 cells. Cells were treated 24 h later with cDDP, PARP1-i, HT and IR. After treatment, the medium was refreshed for medium supplemented with Annexin V (AdipoGen Life Sciences, San Diego, CA, USA) and YoYo3 (Fisher Scientific, Waltham, MA, USA) to visualize early apoptotic (Annexin V-positive) and late apoptotic/necrotic (YoYo3-positive) cells. The 24-wells plate was placed on the microscope stage in the incubator chamber in 5% CO₂ at 37 °C. The Images were automatically acquired at different time intervals for 6 days. The data were processed using IncuCyte image analysis software (IncuCyte S3 Software, Essen BioScience Ann Arbor, MI, USA).

Cells were seeded in flat-bottom 24-well plates 24 h prior to the start of treatments. Next, 24 h after treatment, the medium was replaced by supplemented FluoroBrite DMEM containing 1 μg/mL Annexin V (Adipogen, San Diego, CA, USA) and 200 nM YOYO3 (Thermo Fisher, Waltham, MA, USA). Plate lids were replaced with Breathe-Easy^® sealing membrane (Thermo Fisher) to minimize evaporation. Imaging and quantification were done using the IncuCyte S3 imaging platform (Sartorius, Göttingen, Germany). Phase contrast images were used to quantify cell surface area as a readout for growth. To estimate apoptosis (indicated as “apoptotic material”), the integrated fluorescence intensity of Annexin V⁺/YOYO3⁺ regions was normalized to the cell surface area.