Fig. 3

MMP9 and VEGFA mediate HHLA2-promoted invasion and tumor angiogenesis, respectively. (A) Western blot analysis of the indicated proteins in LO2 cells cotransfected with HHLA2 and c-MET (wild-type or kinase-dead) in the combinations shown. Western blots shown are representative of three independent experiments; relative quantification values are indicated below the bands, and full quantitative data/statistical analysis are provided in the supplementary Excel spreadsheet. (B) qRT-PCR analysis of MMP9 mRNA expression in HepG2 cells with or without HHLA2 expression. (C) Transwell invasion assays of HepG2-Vec and HepG2-HHLA2 cells with or without MMP9 knockdown. (D, E) VEGFA mRNA expression assessed by qRT-PCR (D) and VEGFA secretion assessed by ELISA (E) in HepG2-Vec and HepG2-HHLA2 cells. (F, G) HUVEC tube formation assays using conditioned media from HepG2-Vec or HepG2-HHLA2 cells, with or without VEGFA depletion using an anti-VEGFA antibody (F) or in the presence or absence of 50 ng/ml apatinib (VEGF receptor inhibitor) (G). P values were determined by two-tailed Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bars, 100 μm