Fig 2: Suppressive effects of topiroxostat, a selective XOR inhibitor, on neointima formation induced by carotid artery ligation in NASH mice.(A) Male C57BL/6J mice were divided into 3 groups: fed NC or fed a CDAHFD treated with or without TPX (1 mg/kg/d). Unilateral carotid artery ligation was performed to induce neointima formation 5 weeks after feeding with each diet. TPX was administered from 1 week prior to carotid artery ligation, and all mice were examined 3 weeks after surgery. (B) Serum AST and ALT in each group. n = 11 for NC and n = 12 for CDAHFD/TPX (-) and CDAHFD/TPX (+). (C) Plasma XOR activity measured by the LC/TQMS method in each group. n = 3 for NC and n = 12 for CDAHFD/TPX (-) and CDAHFD/TPX (+). (D) Plasma concentrations of HX, Xan, and UA in each group. n = 3 for NC and n = 12 for CDAHFD/TPX (-) and CDAHFD/TPX (+). (E) Representative H&E-stained sections of ligated carotid arteries obtained from each group. Scale bars: 200 µm. (F) Quantification of the intima/media ratio at 600 µm, 800 µm, and 1000 µm from proximal to the ligated site. n = 11 for NC and n = 12 for CDAHFD/TPX (-) and CDAHFD/TPX (+). Data are shown as mean ± SEM; **P < 0.01 and ***P < 0.001 vs. NC and ††P < 0.01, and †††P < 0.001. NS (1-way ANOVA with Tukey’s post hoc test). I/M, intima/media.

Fig 3: Significant reductions in plasma XOR activity after bariatric surgery and correlations with liver transaminases.Each clinical parameter was examined in 12 obese patients before (Pre) bariatric surgery and 1 week and 1 year after bariatric surgery. Plasma XOR activity was measured using the LC/TQMS method. (A) Serial changes in BMI, plasma XOR activity, serum AST, and ALT after the surgery. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. Pre (paired t test). (B and C) Pearson’s correlation coefficients between changes in plasma XOR activity and those in serum AST, ALT, and BMI at 1 week (B) and 1 year (C) after the surgery. XOR, xanthine oxidoreductase; LC/TQMS, triple quadrupole mass spectrometry and liquid chromatography; AST, aspartate aminotransferase; ALT, alanine aminotransferase.

Fig 4: XDH knockdown increases cell migration and invasion in HepG2 cells. (a) Western blot analysis of XDH expression in HCC cell lines. The band intensities were quantified by ImageJ software. (b) Western blot analysis of XDH knockdown efficiency and quantification of XDH activity in HepG2 cells after shRNA transfection. (c, d) Scratch assay (c) and transwell assay (d) of the migration and invasion of HepG2 cells transfected with control shRNA (shCtrl) or shRNA against XDH (shXDH). Coverage percentages were determined, and quantitative analyses of the numbers of invading cells in each group were performed. Scale bar, 100 µm. (e, f) mRNA profiling (e) and western blot analysis (f) of EMT marker gene expression levels in HepG2 cells transfected with shRNA. XDH, xanthine dehydrogenase; EMT, epithelial-to-mesenchymal transition; mRNA, messenger RNA; HCC, hepatocellular carcinoma; shRNA, small-hairpin RNA; rel., relative. All data are expressed as the mean±s.e.m. of three experiments. Unpaired t-tests were performed to assess statistical significance. ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig 5: XDH overexpression decreases cell motility and invasion in MHCC97H cells. (a) XDH overexpression in MHCC97H cells was confirmed by western blot analysis. The decreases in cell migration were demonstrated by corresponding decreases in coverage percentages. (b) Quantitative analysis of the numbers of invading cells in MHCC97H cells with XDH overexpression or cells treated with control vectors. (c, d) Gene profiling (c) and western blot analysis (d) of EMT marker gene expression levels in MHCC97H cells transfected with cDNA. (e) qRT–PCR analysis of TGFß isoform transcript expression in MHCC97H cells. rel., relative. (f) Western blot analysis of TGFß isoform and Smad2/3 phosphorylation levels in MHCC97H cells. Quantitative analysis of protein expression levels was performed using ImageJ software. (g) Representative photograph of a hematoxylin–eosin-stained lung section in nude mice after tail vein injection of MHCC97H cells. Black arrows indicate the metastatic tumor foci in the lungs. Scale bar, 100 µm. n=8 for each group. Quantitative analyses of the numbers of nodules per lung were performed. XDH, xanthine dehydrogenase; EMT, epithelial-mesenchymal transition. Unpaired t-tests were performed to assess statistical significance. All in vitro data are expressed as the mean±s.e.m. of three experiments. XDH, xanthine dehydrogenase; EMT, epithelial-mesenchymal transition; qRT–PCR, quantitative reverse transcription polymerase chain reaction; HCC, hepatocellular carcinoma; TGFß, transforming growth factor beta; Smad, mothers against decapentaplegic, drosophila. ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.