Insulin

Normal human epidermal keratinocytes (NHKCs; Kurabo, Osaka, Japan) were cultured in HuMedia-KG supplemented with insulin (10 μg/mL), hEGF (0.1 ng/mL), hydrocortisone (0.5 μg/mL), gentamicin (50 μg/mL), amphotericin B (50 μg/mL), and fetal bovine serum (0.4% v/v; Kurabo). Cells were cultured at 37°C in a 5% CO₂ atmosphere. Upon reaching 80% confluence, cells were treated with FICZ (10 nM), or DMBA (1 μM) for 4 h.

Normal human epidermal keratinocytes were purchased from Kurabo (Osaka, Japan). To compare the response of keratinocytes from newborn babies and adults, four different batches of newborn keratinocytes vials from different donors (0 years–1, 2, 3 and 4) and two different batches of adult keratinocytes vials (donors aged 40 years and 57 years) were used (details are summarized in Supplementary Table 1). Cells were cultured in EPILIFE^TM medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with insulin (10 μg mL⁻¹), human recombinant epidermal growth factor (0.1 ng mL⁻¹), hydrocortisone (0.67 μg mL⁻¹), gentamicin (50 μg mL⁻¹), amphotericin B (50 ng mL⁻¹), and bovine pituitary extract (0.4%, v/v), all of which were sourced from Kurabo, at 37 °C in a CO₂ incubator. Undifferentiated keratinocytes between passage 2 and passage 6 were used for experiments. EPILIFE^TM medium contains Mg²⁺, but the concentration is not disclosed.
For fluorescence imaging, keratinocytes were seeded on glass bottom dishes (IWAKI, Shizuoka, Japan) coated with 5 μg mL⁻¹ collagen (Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 6–8 × 10⁴ cells mL⁻¹.