Fig. 3
The relationship between PBOV1 expression level and Hep3B cell growth/metastasis property in vitro. a Hep3B cell growth rates determined by MTT assay. b Hep3B cell cycle analysis by flow cytometry assay. c Cell proliferation reflected by anchorage-independent growth assay (first panel, top down; Scale bar, 1 mm), cell colony formation assay (second panel, top down; Scale bar, 10 mm), and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL) cell apoptosis assay (third panel, top down; Scale bar, 20 μm) (assay; Blue, DAPI-indicating nuclei; green, FITC indicating apoptotic nuclei). d Protein expression levels of survivin, cyclin D1, and cleaved-caspase 3 determined by western blotting assay. e Tube formation assay by human liver tumor endothelial cells (SK-Hep1) cultured on Matrigel-coated plates with conditioned media collected from the Hep3B cell incubation (first panel, top down; Scale bar, 100 μm). Hep3B cell migration ability determined by transwell migration assay (second panel, top down; Scale bar, 50 μm). Hep3B cell invasion ability determined by transwell invasion assay (third panel, top down; Scale bar, 50 μm). Hep3B cell mobility measured by testing the rate of wound healing at 24 h after serum starvation and wound creation (fourth panel, top down; Scale bar, 500 μm) (See also Supplementary Fig. 5b). f Protein expression levels of angiogenesis marker HIF-1α, epithelial markers α-Catenin and E-cadherin, and mesenchymal markers N-cadherin and Vimentin determined by western blotting assay. g Immunofluorescence assays for epithelial markers α-Catenin and E-cadherin, and mesenchymal markers N-cadherin and Vimentin. Scale bar, 20 μm (Blue, DAPI-indicating nuclei; green, FITC indicating α-Catenin or N-cadherin protein; red, Cy5 indicating E-cadherin or Vimentin protein). (See also Supplementary Fig. 5d). Data were represented as means ± S.D. Error bars represent the S.D. values (n = 3; *P < 0.05; ns, not significant. Significant differences were analyzed by two-way ANOVA with multiple comparisons for (a); Student’s t test for (a) and (b), compared with PBS control group)
