Timp 2

Medium was concentrated using StrataClean resin (Agilent Technologies Ltd) and processed for western blotting as previously described [13 (link)] using antibodies to chemerin (R&D Systems), TIMP-1, TIMP-2 and MMP-1 (R&D Systems).

Scratch wound migration assays were performed on confluent monolayers of AGS cells as previously described [41 (link)]. Transwell migration and invasion chemotaxis assays were performed using BD inserts (Corning, NY, USA) as previous described (25,000 cells per insert) [46 (link)]. Chemerin or CAM-conditioned medium (CM) were added in the lower well together with CCX832, α-NETA, Ro-320432, human recombinant TIMP-1 or TIMP-2 (R&D Systems), or vehicle, as appropriate.

The concentrations of NGAL, KIM-1, TIMP2 (all from R&D Systems) and IGFBP7 (Cusabio Technology LLC) were measured by using ELISA kits according to the manufacturers’ instructions.

To prepare the protein extracts, the cells were rinsed twice with ice-cold PBS and harvested. After centrifugation, the cells were resuspended and extracted in lysis buffer (Thermo Fisher Scientific, Inc.) for 30 min on ice. Protein concentrations were assayed using Pierce Coomassie Plus reagent according to the manufacturer’s instructions, and 40 μg of protein was loaded for separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to polyvinylidene difluoride membranes (Immobilon-P; EMD Millipore Corporation, Billerica, MA, USA). The membranes were blocked in Tris-buffered saline containing 5 % bovine serum antigen (BSA) and probed with HIF-1a, tissue inhibitor of metalloproteinase-2 (TIMP-2), VEGF-A, hepatocyte growth factor (HGF), matrix metalloproteinase (MMP)-2, and MMP-9 (all from R&D Systems Inc.) corresponding antibodies. Reacted bands were detected by horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence substrates (PerkinElmer, Boston, MA, USA).

Metalloprotease inhibitors, including BB94 (batimastat), TAPI-1 and TAPI-2 (Santa Cruz Biotechnology), and TIMP1, TIMP2, TIMP3, and TIMP4 (R&D Systems), were added at the time of GPR37L1 induction (or 5 h after transfection in the case of U87 MG cells) or 24 h after induction for TX14A experiments, and cells were then incubated for 24 h before lysis. For MG132 proteasome inhibition, cells were induced with doxycycline overnight and then treated with MG132 for 6 h before harvest. Verification of TIMP activity in vitro using activated MMP-2 and the fluorogenic peptide substrate MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH₂ was performed as per the protocol provided by R&D Systems.

The endometrial tissue was mixed with lysis buffer and the protease inhibitor. Then, the samples were homogenized and centrifuged (12,000 rpm × 10 min), and the supernatants were collected. After determining the protein concentration, the samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated with the primary antibody overnight at 4 °C. The antibodies included anti-estrogen receptor α (ERα, Santa Cruz Biotechnology, USA), anti-progesterone receptor A (PRA, Proteintech, China), anti-hypoxia inducible factor 1α (HIF1α, R&D, USA), anti-VEGFA (Proteintech, China), anti-angiopoietin 2 (ANGPT2, Abcam, USA), anti-cyclooxygenase 2 (COX2, CST, USA), anti-prostaglandin E receptor 2 (EP2 receptor, Abcam, USA), anti-matrix metallopeptidase 2 (MMP2, Abcam, USA), anti-MMP9 (Abclone, China), anti-tissue inhibitor of metalloproteinase 2 (TIMP2, R&D, USA), anti-FGF2 (Santa Cruz Biotechnology, USA), and anti-β-actin (Proteintech, China). Next, the PVDF membranes were incubated with the fluorescent secondary antibodies (CST, USA) at room temperature for 1 h. Lastly, the protein bands were scanned using the Odyssey Infrared Imaging System (Li-Cor Biosciences, USA).