Mouse hgf

For in vitro assays, NZ001 was obtained from Nanjing Zhongrunyuan Pharmaceutical Company (Nanjing, Jiangsu, China). XL184, sorafenib and PF-04217903 were purchased from Selleck Chemicals (Houston, TX, USA). Goat anti-mouse VEGF antibody (AF-493-NA) was purchased from R&D Systems (Minneapolis, MN, USA). NZ001, XL184 and PF-04217903 were prepared as a 20-mM stock solution in DMSO (Sigma-Aldrich, St. Louis, USA) for in vitro studies. For in vivo studies, NZ001 was formulated in sterile ddH2O and administered via oral gavage at 10 mg/kg or 30 mg/kg. sorafenib was dissolved in Cremophor EL/ethanol (50:50; Sigma Cremophor EL, 95% ethyl alcohol) at 4-fold (4×) the highest dose. The final dosing solutions were prepared on the day of use by diluting to 1× with ddH2O and were administered via oral gavage at 30 mg/kg. VEGF antibody was injected intraperitoneally 3 times per week at 7.5 mg/kg. Recombinant human HGF, mouse HGF and human VEGF were obtained from R&D Systems (Minneapolis, MN, USA). All information of primary antibodies used for Western blot and immunoprecipitation were shown on Additional file 1: Table S1. All secondary antibodies were purchased from Jackson ImmunoResearch (Philadelphia, PA, USA).

P19.CL6 cells differentiated to cardiomyocytes (7.4 × 10⁴ cells/100 µL in 96-well plate) were incubated at 37 °C overnight, and the medium was replaced with fresh MEMα + 10% FCS just before the addition of recombinant mouse Reg IV protein (R&D Systems) or mouse Hgf (R&D Systems). After a 24-h treatment with Reg IV or Hgf, the viable cell numbers were determined by a Cell Counting kit-8 (Dojindo Laboratories, Mashiki-machi, Japan), according to the manufacturer’s instructions. Briefly, WST-8 solution was added to cells in 96-well plates, and the cells were incubated at 37 °C for 30 min. The optical density of each well was read at 450 nm (reference wave length at 650 nm) using a Sunrise^TM microplate reader (Tecan, Männedorf, Switzerland), as described [36 (link),58 (link),75 (link),79 (link),85 (link),108 (link)].