Coating matrix

Thawed keratinocytes at passage 2 were seeded 1.84 × 10⁴/cm² in 2 wells of a 4-well dish coated with Coating Matrix (Gibco). Cells were reprogrammed 2 days post-seeding, using the CytoTune-iPS 2.0 Sendai Virus Reprogramming Kit (ThermoFisher) according to the manufacturer’s instructions, with modifications (Re et al., 2018 ). Cells cultured in EpiLife supplemented with HKGS and 5 mM Y-27362 were transduced with the viral vectors at MOIs reported in (Re et al., 2018 ) and placed in a humidified incubator at 37°C, 5% CO₂. The next day the media was replaced with media without viral vectors. Six days post-infection cells were passaged onto previously inactivated MEFs and transferred to a hypoxic incubator at 37 °C, 5% CO2, 5% O2. The medium was progressively switched over 4 days from day 13 to 100% KSR medium (Advanced DMEM(Gibco)/Knock-Out Serum Replacer (Gibco)/Glutamax (Gibco)/2-Mercaptoethanol (Gibco)/4ng/ml FGF 2 (Gibco)) with 10µM Y-27362.
After the emergence of iPSC-like colonies, those with appropriate morphology were manually picked on day 28 and transferred to Matrigel coated 6-well plates with mTeSR1 medium (StemCell Technologies) containing 10µM Y-27362. Medium was changed after 24 hours. Colonies were expanded by splitting at 1:3 to 1:6 ratio every 4-6 days and maintained in a hypoxic incubator at 37 °C, 5% CO2, 5% O2.

All the cell lines were purchased from American Type Culture Collection. HaCaT, HEK293T, SiHa, C33A, and HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco), and HT-3 and U2OS cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) in the presence of penicillin, streptomycin (Gibco) at 37 °C in humidified atmosphere containing 5% CO₂.
Primary human cervix keratinocytes (PHKs) were isolated from normal cervical epithelial obtained from iCell (HUM-iCell-f016). PHKs were cultured in EpiLife Medium (Thermo Fisher, MEPI500CA) with the addition of EpiLife Defined Growth Supplement (EDGS) (Thermo Fisher, S0125). The Coating Matrix (Thermo Fisher, R011K) was used to enhance the attachment, growth, and population doubling potential of human keratinocytes. For all the experiments, keratinocytes cultured between the third and fifth passages were used.
For H₂O₂ treatment and NAC supplement, cells were plated in complete culture media with 10 μM H₂O₂ for 6 h, then exchanged the medium supplemented with 1 mM NAC for another 6 h.

Eight‐well chamber slides (VWR International, Radnor, PA, USA) were coated with coating matrix (Thermo Fisher Scientific, Waltham, MA, USA), then NHEKs were seeded (50,000‐ 100,000 cells/well) in 500 μl cell culture medium. For staining the cells were fixed with 100 μl/well 4% formaldehyde in PBS for 5 min, then washed and permeabilized with 0.1% Triton x‐100, 100 μl per well, for 5 min. For blocking the permeabilized cells were incubated for 1 h with 5% FBS, 2% BSA in PBS on shaker. After blocking 200 μl of the primary Abs were added (1:100 in PBS) and incubated at RT 1 h with gentle shaking. NucBlue (Thermo Fisher Scientific, Waltham, MA, USA) was diluted in PBS (2 drops per ml) and the secondary Abs were diluted in NucBlue/PBS at 1:200. 200 μl of the diluted secondary antibodies were added and incubated 30 min at RT with gentle shaking. Samples were coverslipped with ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) and images acquired on the Leica TCS SP8 x microscope (Leica Microsystems, Wetzlar, Germany).