Fig. 3

RNF144A-AS1 accelerated tumor metastasis, angiogenesis and growth in vivo. A Western bolt analysis of the expression of EMT markers and VEGFA in treated GC cells as indicated. B qRT-PCR analysis of the expression of EMT markers in treated GC cells as indicated. C Expression level of RNF144A-AS1 in MKN45 and AGS cells transfected with RNF144A-AS1-targeting shRNA or control. D, E Images of HE staining to lung sections with metastatic sites and the number of lung metastatic nodules to each group using treated MKN45 (D) or AGS (E) cell lines. Scale bars = 200 μm. F Representative images showing Matrigel plugs for RNF144A-AS1 knockdown group and control group (left panel) and representative images of skin vasculature adjoining indicated plugs (right panel). G IHC analysis of CD31 in indicated plug sections and the microvascular density (MVD) value was calculated for each group. Scale bars = 100 μm. H Representative images of Matrigel plugs and adjoining skin vasculature using treated AGS cells. I IHC analysis of CD31 and quantification of the microvascular density from indicated plug sections. Scale bars = 100 μm. J and L Xenograft tumors from MKN45 (J) and AGS (L) cells were harvest from nude mice (left panel), and the growth curve (middle panel) and final tumor weight (right panel) were measured. K and M Images of Ki67 staining for xenograft tumor sections from MKN45 (K) or AGS (M) cells. Scale bars = 50 μm. Error bars, mean ± SD from triplicate samples. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t‐test unless otherwise specified