Nunc lab tek 8 well coverslips

Spheroids were transfected with CPP NPs with Cy5 fluorescently labelled siRNA, and then 1.5 h later washed with regular culture media and transferred to Nunc Lab-Tek 8-well coverslips (Thermo Fisher Scientific, Waltham, MA, USA) using 1 mL ultra-low-retention pipette tips (Brand, Wertheim, Germany). Spheroids were kept at 37 °C in a 5% CO₂ environment during the whole experiment and live-imaged under LSM710 confocal microscope (Zeiss, Oberkochen, Germany) at different time points (2, 4, and 6 h). Z-stack acquisition was used and mid-height section images are presented. Images were analyzed using ZEN microscope software (Zeiss, Oberkochen, Germany).

U-87MG tumor and endometriotic primary cells were seeded on Nunc Lab-Tek 8-well coverslips (Thermo Fisher Scientific, Waltham, MA, USA) and when they had become about 50% confluent, cells were transfected with CPP/siRNA NPs. The cells were fixed with 4% formaldehyde (Naxo, Tartu, Estonia) 48 h post-transfection. Nonspecific staining was reduced using species-specific blocking solution containing 0.25% TritonX-100 in PBS supplemented with 5% normal donkey serum (Sigma-Aldrich, St. Louis, MO, USA). Cells were incubated overnight with primary R2 antibody (#sc-10846, Santa Cruz, Dallas, TX, USA) against RRM2 in blocking solution (1:500), followed by 3 h incubation with Alexa488-labelled secondary antibody (#A-11055, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) in blocking solution (1:1000). Nuclei were counterstained with DAPI (Sigma-Aldrich, St. Louis, MO, USA). Cells were visualized using an LSM710 confocal microscope and images were analyzed using ZEN microscope software (Zeiss, Oberkochen, Germany).

Migration assay was adapted from previously described protocols [21 (link),22 (link)]. In more detail, spheroids were transfected with different CPP/siRNA NPs 4 days after seeding. Then, 48 h after transfection, spheroids were transferred to the middle of Nunc Lab-Tek 8-well coverslips (Thermo Fisher Scientific, Waltham, MA, USA) previously coated with 0.1% gelatin (Naxo, Tartu, Estonia), one spheroid per well. A further 3 days after that, 3D cultures were fixed with 4% formaldehyde (Naxo, Tartu, Estonia) and stained with 1% methylene blue (BioGnost, Zagreb, Croatia) and 1% eosin (BioGnost, Zagreb, Croatia). Spheroids were visualized and photographed under stereomicroscope (Leica M165FC, Wetzlar, Germany). Images were analyzed and cell migration surface area was measured using ImageJ software.
Migration evaluation was the only assay, where HT1080 cells were used instead of U-87MG due to technical reasons. Namely, U-87MG cell migration from the spheroids was scattered and irregular, which did not allow the quantification of the results. Therefore, the U-87MG cell line was replaced with another 3D model available that had previously shown to exhibit regular cell migration patterns according to our experience.