Yoyo 3

To determine whether coating of VV was successful, flow virometry of VV coated with Chol-PEG(10K)-FITC (named FITC-PCVV) was performed.
YOYO-3 (Thermo Fisher Scientific, Y3606) was used for nucleic acid staining of VV.¹³ (link)^,44 VV was incubated with 1 μM YOYO-3 at room temperature for 20 min. The sample was then centrifuged at 20,000 × g for 10 min. The viral pellet was washed three times in PBS as described in section 4.1. Final virus pellets were either resuspended in PBS to a concentration of 5.3 × 10⁵ virus particles (VG)/μL and further modified to form PCVV, or 2.6 × 10⁵ VG/μL as the naked form.⁴⁴ Stained VV virions further modified to PCVV were coated and purified as detailed in section 4.1, and resuspended at a final concentration of 2.6 × 10⁵ VG/μL. An Attune NxT cytometer (Invitrogen) was adapted to record FSC and SSC through the violet channel using the Attune NxT No-Wash No-Lyse filter kit (Thermo Fisher Scientific, 100022776). VV populations were gated for positive YOYO-3 staining. Analysis was performed using FlowJo v10.6.1 software. FITC-PCVV-YOYO-3 and VV-YOYO-3 were gated for positive YOYO-3 and FITC staining.

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).