Irigenin

Six-week-old male wild-type C57BL/6 mice were purchased from the Shandong University Laboratory Animal Centre. For hepatic fibrosis model, mice were administered CCl₄ (2 ml/kg, CCl4: olive oil = 1:4) or olive oil twice a week by intraperitoneal injection. AAV9-FN-EDA-shRNA or AAV9-Ctrl (Genechem, Shanghai, China) were injected into mice through tail vein every four weeks for eight weeks. Irigenin (5 mg/kg, Selleck, Shanghai, China) or water was administered through gavage after two weeks CCl₄ administration. The animal study protocol was approved by the Ethics Committee of Shandong Provincial Hospital.

The tube formation assay was performed as previously described^{8 (link)}. Briefly, endothelial cells were seeded at a density of 1–2 × 10⁴ cells/well for 6 h in plates precoated with Matrigel at 37 °C and 5% CO₂. For coculturing experiment, LX-2 cells were seeded into the upper chambers of a transwell plate (Corning, Costar 3422) for 24 h and then transferred to other plates with endothelial cells. Experiments were performed in the presence or absence of various fibronectins, including pFN (40 μg/ml, ECM001, Sigma-Aldrich; St. Louis, MO, USA), FN-EDA (40 μg/ml, F2518, Sigma-Aldrich; St. Louis, MO, USA), rEDA (40 μg/ml Flag-tagged, Daian, Wuhan, China) or inhibitors including irigenin (5 μM Selleck, Shanghai, China) and, resatorvid (5 μM, MCE, USA), or neutralizing antibodies (Table 4). Tube formation was photographed using inverted microscope and quantified by calculating the average tube length using ImageJ software (National Institutes of Health, Bethesda, MD, USA)^{58 (link)}.