Hydrocortisone

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.

The human colon cancer cell lines HT29, CaCo-2, DLD-1 and Colo320 were obtained from American Type Culture Collection (Rockville, MD). DLD-1 and CaCo-2 were maintained in microscale essential medium eagle (Sigma Chemical Co., St. Louis, MO) with high glucose and 10% fetal bovine serum (FBS). HT-29 was cultured in McCoy’s supplemented with 10% FBS. Colo320 was maintained in RPMI-1640 medium (Sigma Chemical Co.) supplemented with 10% FBS. HUVECs were obtained from Kurabo Co. (Osaka, Japan). HUVECs were maintained in HuMedia-EG2 medium supplemented with 2% FBS, 5 ng/ml basic fibroblast growth factor, 10 μg/ml heparin, 10 ng/ml epidermal growth factor and 1 μg/ml of hydrocortisone according to the supplier’s instruction (Kurabo Co.). All cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air.