Bovine pituitary extract

The primary culture of human epidermal keratinocytes was kindly supported by J-TEC (Gamagori, Japan). Keratinocytes were isolated from 3 patients with each of the 3 types of SNPs in AKR1C3; rs12529 GG and rs12387 AA, rs12529 GC and rs12387 AG, and rs12529 CC and rs12387 GG. Skin tissue obtained from the patients was isolated as individual cells and proliferated according to a protocol using 3T3 cells as feeders^{36 (link)}. The completed cell lines were incubated in HuMedia-KG2 medium supplemented with 0.1% insulin, 0.1% human epidermal growth factor, 0.1% hydrocortisone, 0.4% bovine pituitary extract, and 0.1% gentamicin/amphotericin B (Kurabo, Osaka, Japan).

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 synthetic substrate for elastase, N-succinyl-tri-alanyl-p-nitroaniline (STANA) was purchased from the Peptide Institute Inc (Osaka, Japan). The anti-human NEP (neutral endopeptidase) antibody was purchased from Santa Cruz Biotechnology (CA, USA). Rabbit anti-human MMP-1 (Collagenase Type1) rabbit antibody was purchased from Sigma (MO, USA). Horseradish peroxidase conjugated goat polyclonal anti-mouse IgG was obtained from Transduction Laboratories (NJ, USA), ELISA kits (for IL-1α, IL-1ß, IL-6, IL-8, GM-CSF and endothelin (EDN)-1) were obtained from Endogen (Thermo Fisher Scientific, Yokohama, Japan). Ubiquinol₁₀ was kindly provided by Kaneka (Kaneka QH^™ from Kaneka) via Dr. YY. Human primary keratinocytes (HPKs), human dermal fibroblasts (HDFs), serum-free keratinocyte growth medium (Medium 154S) containing low calcium (0.2 mM), bovine pituitary extract (BPE) and epidermal growth factor (EGF), were obtained from Kurabo (Tokyo, Japan). HaCaT human keratinocytes were kindly supplied by Dr. M. Furue (Kyushu University, School of Medicine, Department of Dermatology). Recombinant human GM-CSF (215-GM-010) was purchased from R&D Systems (MN, USA). Recombinant human IL-1α (200-01A) was obtained from PEPROTECH (London, UK). Other chemicals of reagent grade were purchased from Sigma-Aldrich (MO, USA).

Serum-free keratinocyte growth medium (Medium 154S) containing low calcium (0.2 mM), bovine pituitary extract (BPE) and epidermal growth factor (EGF) were obtained from Kurabo (Tokyo, Japan). Human primary keratinocytes (HPKs) were purchased from Biopredic Intl (Saint-Gregoire, France). Human HaCaT keratinocytes [22 (link), 23 (link)] were kindly supplied by Dr. M. Furue (Kyushu University, School of Medicine, Department of Dermatology). Rabbit polyclonal anti-TGase 1 (NOV-1184-45: NB100-1844 Lot7E3) was purchased from Novus Biologicals (Littleton, CO, USA). Antibodies to phospho-ERK, ERK, phospho-p38, p38, phospho-JNK, JNK, phospho-AKT and AKT were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies to phospho-Ser468/Ser276/Ser536NFκBp65, NFκBp65, phospho-Ser133CREB, CREB, c-Jun, phospho-Ser73c-Jun, c-Fos, ATF-2, phospho-Thr71ATF-2, MSK1 and phospho-Thr581/Ser 376/Ser360MSK1 were also purchased from Cell Signaling Technology. Antibodies to COX-2 and β-actin were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). The NFκB activation inhibitor II (JSH-23), the MEK inhibitor (PD98059), the p38 inhibitor (SB203580), the JNK inhibitor (JNK inhibitor II) and H89 were purchased from Calbiochem (San Diego, CA, USA). Other chemicals including AX were obtained from Sigma-Aldrich Corp.