12 and 24 well plates

Cryopreserved PHH from five different donors (Table 1) were commercially acquired from BioIVT (Maryland, US). The supplier sought informed consent from each prospective donor or the subject's legally authorized representative and these forms, along with their corresponding protocols, were reviewed and approved by the appropriate regulatory and ethics authorities in accordance with HHS regulations for the protection of human subjects (45 CFR §46.116 and §46.117) and Good Clinical Practice (ICH E6). Cells were thawed and seeded in PHH culture medium as previously described.^[14] For 2D PHH culture, cells were seeded at 3,5×10⁵ cells per mL onto 12 and 24‐well plates (Corning) coated with rat tail collagen I (Corning) in PHH culture medium. PHH were allowed to attach for 2 h, after which the medium was replaced with serum‐free PHH culture medium. Cells were maintained for 7 days with medium change every 48–72 h. For Wnt signaling activation, PHH spheroids were seeded in PHH culture medium containing 3 µm CHIR99021 (Tocris). For EdU DNA synthesis labeling, EdU was incorporated into the culture medium at 10 µm from seeding and EdU incorporation rates were assessed after 7 days.

HeLa cells were obtained from the laboratory of Lars Jansen at Instituto Gulbenkian de Ciência (IGC) and maintained in Dulbecco's modified Eagle medium high glucose with L-glutamine and sodium pyruvate (Biowest) supplemented with 10% fetal heat-inactivated (Biowest) and 1% penicillin–streptomycin (Biowest)—Dulbecco's modified Eagle medium complete medium. Cells were maintained in humidified atmosphere, 5% CO₂ at 37 °C. Cells were counted in a haemocytometer (Neubauer Cell with Double Grid, Fisher Scientific) using 1% trypan blue solution (Sigma-Aldrich) and plated at 10⁵ and 6 × 10⁴ cells per well in 12- and 24-well plates (CORNING), respectively.
All incubations used a concentration of 6.50 nM of DT and PEGyDT. Different time points (6–72 h) were prepared for FACS analysis. The medium and cells were collected and cells were centrifuged for 4 min, 3,250g, at 4 °C. Cells were resuspended in 150 μl FACS buffer (2% FBS, 0.02% sodium azide-1-¹⁵N (Sigma-Aldrich) in PBS 1x) and 25 μl propidium iodide (Sigma-Aldrich) were added.
Data were acquired in a Becton Dickinson Flow Cytometer FACSCalibur. Data were acquired using CellQuest software (Becton Dickinson) and analysed in FlowJo.