Cox 2

After isolating total proteins from HESCs or nucleus with the lysis buffer, a BCA kit (Abcam, Cambridge, UK) was utilized to determine the concentration of proteins, followed by loading the proteins into the 12% SDS-PAGE. After separation, proteins in the gel were transferred onto the PVDF membrane (Abcam, Cambridge, UK), followed by incubation in the TBST buffer containing the primary antibody against COX-2 (1:1000, GeneTex, Texas, USA), p-p38 (1:2000, GeneTex, Texas, USA), p-IκBα (1:500, GeneTex, Texas, USA), IκBα (1:3000, GeneTex, Texas, USA), NF-κB p65 (1:3000, GeneTex, Texas, USA), or Tubulin (1:8000, GeneTex, Texas, USA). After incubating at 4°C overnight, the membrane was incubated with the secondary antibody (1:2000, GeneTex, Texas, USA) at room temperature for 90 min. Lastly, the ECL solution was utilized to expose the membrane and the expression of proteins was quantified using the Image J software [13 (link)].

Animal experimentation was conducted according to approved standards for animal care by the Institutional Review Board of Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine. C57BL/6 mice (Charles River, Beijing, China) aging from 10 to 13 weeks were mated overnight. The presence of a vaginal plug in the morning was counted as gestational day 0.5. Fetal membranes were collected on gestational days 14.5 and 18.5, and fixed for the study of the distribution of C/EBPδ, COX‐2, and 11β‐HSD1 with immunohistochemical staining of paraffin‐embedded tissue sections using antibodies against C/EBPδ (GeneTex), COX‐2 (Cell Signaling Technology), and 11β‐HSD1 (Abcam). To study gestational changes of C/EBPδ, COX‐2, and 11β‐HSD1, fetal membranes were collected on gestational days 14.5, 15.5, 16.5, 17.5, and 18.5 for protein extraction and measurements of C/EBPδ, COX‐2, and 11β‐HSD1 abundance with Western blotting.

Cisplatin, AMF, wortmannin, and other reagents were acquired from Sigma-Aldrich (St. Louis, MO, USA). Creatinine (CRE) and blood urea nitrogen (BUN) detection kits were available from HUMAN Diagnostics Worldwide (Wiesbaden, Germany). The serum levels of cytokines IL-1β, IL-6, and TNF-α were detected with the ELISA MAX Deluxe Kit (BioLegend, San Diego, CA, USA). Antibodies against cyclooxygenase-2 (COX-2) (1:1500), p-JNK (1:1000), catalase (1:2000), superoxidase dismutase 1 (SOD1) (1:1000), SIRT1 (1:1000), AMPK (1:1000), glutathione peroxidase 3 (GPx3) (1:1000), and TLR-4 (1:1000) were provided from GeneTex (San Antonio, TX, USA). Antibodies against JNK (1:2000), p-ERK (1:500), ERK (1:1000), p-p38 (1:1000), p-CaMKK (1:1000), p-AMPK (1:1000), p-PI3K (1:1000), PI3K (1:2000), AKT (1:1500), p-AKT (1:1500), Beclin 1 (1:1000), LC3B (1:1000), P62 (1:1000), and p-IκB-α (1:1000) were bought from Cell Signaling Technology (Beverly, MA, USA). Antibodies that contrapose inducible NO synthase (iNOS) (1:1500), NF-κB (1:1000), IκBα (1:1500), HO-1 (1:2000), Nrf2 (1:1500), p38 (1:1000), and β-actin (1:10,000 dilution) were bought from Abcam (Cambridge, UK). β-actin was used as the loading control.

SAC (purity 98.6%) (Figure 1A) was obtained from Chem Faces Pharmaceutical Company (Wuhan, China). Cisplatin, amifostine (AMF), EX-527 and other solvents and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). BUN and CRE assay kits were supplied by HUMAN Diagnostics Worldwide (Wiesbaden, Germany). ELISA Max TM Set Deluxe kits, used to test IL-1β, IL-6 and TNF-α in mice, were obtained from BioLegend Inc. (San Diego, CA, USA). For Western blotting, primary antibodies that contrapose COX-2, p-JNK, catalase, SOD1, Sirt1, AMPK, GPx3 and TLR-4 were bought from GeneTex (San Antonio, TX, USA). Antibodies against JNK, p-ERK, ERK, p-p38, p-CaMKK, p-AMPK and p-IκB-α were bought from Cell Signaling Technology (Beverly, MA, USA). Antibodies that contrapose iNOS, NF-κB, IκBα, HO-1, Nrf2, p38 and β-actin were bought from Abcam (Cambridge, UK, USA). β-actin is used as an endogenous control protein.