Figure 4

The effects of XDH downregulation on HCC cells are dependent on the TGFβ-Smad signaling pathway. (a) Scratch assay of the migration of HepG2 cells with stable XDH knockdown (shXDH) or control (shCtrl) cells treated with TGFβ pathway inhibitors (GW788388 or pirfenidone) for 48 h. (b) Scratch assay of the migration of HepG2 cells incubated in the presence or absence of 50 μM oxypurinol, 100 μM GW788388 or 2 nM pirfenidone. (c) Quantitative analysis of the numbers of invading cells in the XDH knockdown (shXDH) HepG2 and control (shCtrl) cell populations in the absence or presence of 100 μM GW788388 or 2 nM pirfenidone. (d) Quantitative analysis of the numbers of invading cells in the Huh7 cell population in the absence or presence of 100 μM GW788388, 2 nM pirfenidone or 50 μM oxypurinol. (e, f) Scratch assay of the migration of HepG2 cells (e) or Huh7 cells (f) treated with 50 μM oxypurinol, 5 ng/ml TGFβ1 or a combination of both for 48 h. Quantitative analyses of coverage percentages and western blot analyses and quantification of EMT marker expression levels in each group were performed. Unpaired t-tests were performed to assess statistical significance. All data are expressed as the mean±s.e.m. of three experiments. DMSO, dimethyl sulfoxide; EMT, epithelial-to mesenchymal transition; XDH, xanthine dehydrogenase; qRT–PCR, quantitative reverse transcription polymerase chain reaction; HCC, hepatocellular carcinoma; TGFβ, transforming growth factor beta; Smad, mothers against decapentaplegic, drosophila; shRNA, small-hairpin RNA. ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.