Gremlin

SSD (CAS: 20874-52-6, China) was purchased from Chengdu Purifa Technology Development Co. LTD; NaCMC(C8621-25g, China) was purchased from Beijing Solebao Technology Co. LTD; TGF-β1 (AF-100-21C, United States) was purchased from PeproTech; BMP7 (Recombinant Human Bone Morphogenetic Protein 7, HY-P7008, United States) was purchased from MedChemExpress; and Gremlin (SRP4657, United States) was purchased from Sigma.

Gremlin, RAD001, rapamycin and perifosine were purchased from Sigma Chemicals (Shanghai, China). AZD8055 and OSI-027 were purchased from Selleck (Nanjing, China). All phosphorylation antibodies and their non-phosphorylated controls were obtained from Cell Signaling Tech (Danvers, MA). Anti-VEGF antibody and all other antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Cell signalling inhibitors were applied directly to cortical cultures (1 h before exposure to CM) or indirectly via the in vitro model, the BeWo barriers (at exposure to altered oxygen) to see the effect on CM. Map kinase inhibitor SB203580 (20 μM; InviviGen), epidermal growth factor receptor (EGFR) inhibitor PD168393 (20 μM; Santa Cruz Biotechnology), Noggin (200 ng/ml; StraTech), Gremlin (Gremlin-2, 500 ng/ml; Sigma) and Chordin (chordin-like 1, 500 ng/ml; Sigma) were tested. Inhibitors were chosen to block signals in the Smad (Noggin, Chordin, and Gremlin), mTOr-Akt-PI3k (PD168393) and 38-MAPK (SB203580) pathways in an attempt to isolate which if any signalling pathway was being affected. Where appropriate Bone Morphogenetic Protein (BMP) binding antibody (R&D Mab3552) was applied to cultures at a dose of 10 µg/ml 1 h before exposure to CM.