Hepatocyte growth factor (hgf)

We purchased sheep anti-rabbit antibodies of CD29, CD34, CD44, CD45, and fluorescein isothiocyanate- (FITC-) labeled IgG secondary antibody from eBioscience (San Diego, CA). Sheep polyclonal anti-human antibodies to alpha-fetoprotein (AFP), albumin (ALB) and β-actin, and FITC-labeled IgG secondary antibody were obtained from Sigma (St. Louis, MO). Antibodies to DNMT1 and phosphorylated p38 were obtained from Cell Signaling Technology (Beverly, MA). HGF was purchased from Proteintech (Rocky Hill, NJ). Zebularine was purchased from Berry and Associates (Dexter, MI).
The p38 inhibitor SB203580 was obtained from Merck (Germany). A stock solution of SB203580 was prepared in dimethyl sulfoxide (DMSO). SB203580 was diluted in Dulbecco modified Eagle medium (DMEM; Gibco, Rockville, MD) and was added to cell culture 1 h before stimulation with HGF and kept further during the exposure to this compound. A stock solution of zebularine for cellular assays was prepared in DMSO and then diluted in the optimal medium to the final concentrations. Indocyanine green (ICG) was obtained from Aladdin (Shanghai, China).

For immunohistochemical staining, after routine deparaffinization, rehydration, antigen retrieval, and endogenous peroxidase inactivation, samples were blocked with goat serum. Then, the sections were incubated with primary antibodies overnight at 4°C. Subsequently, the liver section slides were incubated with streptavidin-HRP, and this was followed by detection with DAB substrate. The antibodies were used at the following dilutions: HGF (1 : 100, Proteintech) and VEGF (1 : 100, Proteintech).

A small piece (approximately 10 mm³) of homogenized tumor tissues and cell culture medium was collected and centrifuged at 4 °C for 10 min. Proteins in the supernatant were then extracted using M-PER Mammalian Protein Extraction Reagent. An ELISA was used to measure the protein concentrations of SRPX2 (LifeSpan BioSciences), uPAR (Abcam), and HGF (Proteintech Rosemont, IL, USA). The assays were performed according to the manufacturers’ instructions and in triplicate. The presented data are the means of three independent experiments.