Pdgf b

The following recombinant human proteins were used: interleukin-24 (IL-24), platelet-derived growth factor B (PDGF-B) (R&D Systems) interleukin-1 beta (IL-1β) and transforming growth factor beta-1 (TGF-β1) (Life Technologies). IL-24 was dissolved in sterile phosphate buffered saline (PBS); IL-1β in sterile distilled water; TGF-β1 and PDGF-B in HCl (4 mmol/l).

The NIH-3T3 cells were seeded in a 10-cm Petri dish. At 80% confluence, they were transfected with a combination of DNA and PEI (6 μL PEI/500 ng DNA, PEI stock concentration 0.324 mg/mL, pH 7.5). Two days post transfections, the cells were selected and maintained in medium containing G418 (1.2 mg/mL; Sigma). To confirm the secretion of soluble factors, each cell line was cultured in a Petri dish in a serum-free DMEM F-12 medium. For inducible systems, doxycycline (1 μg/mL) was added. Then, 24 h after seeding, the supernatants were collected and concentrations of secreted soluble factors were determined using qPCR (KGF and LL-37) or an appropriate ELISA kit [EGF, FGF-2, IGF-I, HGF, VEGF-A (Abcam); PDGF-B (R&D Systems); TGF-β1, IL1-Ra (eBioscience)] according to the manufacturer’s instructions. Reporter cell lines (fLuc ON/OFF) were characterized by measuring luciferase expression. The inducibility of the cells was tested by adding doxycycline in concentrations 0.01–10 μg/mL.

Proteins released from endothelial cells were quantified in samples of conditioned medium using ELISA kits for TGF-β1 (R&D Systems, Minneapolis, MN, USA), PDGF-B (R&D Systems), PDGF-C (Cloud-Clone Corp, Wuhan, China), PDGF-D (MyBiosource Inc., San Diego, CA, USA) or CTGF (Cusabio Life Science, Wuhan, China) according to the manufacturers’ protocols. Collagen content in conditioned medium was quantified using the Sircol Collagen Assay Kit (Biocolor Ltd., Carrickfergus, UK) according to the manufacturer’s protocol.