UII increased expression of NADPH oxidase subunits.

<p>(A) The optimum concentration of UII was determined by MTT assay and Brdu assay. After 24 h starvation, cells were treated with different 10⁻¹⁰–10⁻⁶ M UII for 24 h. Cell viability was tested by MTT assay. Cell proliferation was detected by Brdu incorporation. The results are presented as mean ± SEM (n = 6). * <i>P</i><0.05 versus control. (B) Expression of NADPH oxidase subunits were measured by western blotting and normalized against GADPH or differences between membrane fraction and cytosol fraction. After starvation, cells were treated with UII (10⁻⁹ M) for 2 h. Data are presented as mean ± SEM (n = 3). * <i>P</i><0.05 versus control. (C) Immunofluorescence staining detected intracellular protein levels of p47phox upon UII stimulation. After starvation, cells were treated with UII (10⁻⁹ M) for 2 h with or without apocynin (0.5 mM) pretreated for 30 min. The fluorescence signals were captured by fluorescent microscopy.</p

UII-increased phosphorylation of PI3K/Akt and ERK was partially abolished by apocynin.

<p>The protein levels were measured by western blotting and normalized against GADPH. After starvation, cells were treated with UII (10⁻⁹ M) for 2 h. Data are presented as mean ± SEM (n = 6). * <i>P</i><0.05 versus control. # <i>P</i><0.05 versus UII group.</p

ROS accelerated G1/S transition and increased cyclin E/CDK2 expression in WB-F344 cells.

<p>(A) Cell cycle was assessed by flow cytometry. After starvation, cells were treated with UII (10⁻⁹ M) for 12 h, with or without apocynin (0.5 mM) pre-treatment for 30 min. The results are presented as mean ± SEM (n = 5). * <i>P</i><0.05 versus control. # <i>P</i><0.05 versus UII group. (B) Expression of cyclin E and CDK2 was examined by western blotting and normalized against GADPH. Data are presented as mean ± SEM (n = 3). * <i>P</i><0.05 versus control. # <i>P</i><0.05 versus UII group.</p

ROS level and expression of NADPH oxidase were elevated in HCC.

<p>(A) Fresh human HCC tissues and adjacent noncancerous tissues were stained with DHE. The fluorescence signals were captured by fluorescent microscopy and analyzed by Image J software. (B) Malondialdehyde content in human liver tissue was measured by thiobarbituric acid assay. (C) Activity of superoxide dismutase and glutathione peroxidase was measured by glutathione assay. (D) Expression of NADPH oxidase subunit p47phox, NOX2, p40phox and p67phox was measured by western blotting and normalized against GADPH. The results are presented as mean ± SEM. * indicates significant difference compared to adjacent liver tissues, <i>P</i><0.05.</p

UII-induced ROS production contributes to cell proliferation.

<p>(A) Cell proliferation was determined by Brdu incorporation assay. After starvation, cells were treated with UII (10⁻⁹ M) for 24 h, with or without apocynin (0.5 mM) pre-treatment for 30 min. Data are presented as mean ± SEM (n = 6). *<i>P</i><0.05 versus control; # <i>P</i> < 0.05 versus UII group. (B) The proportion of Brdu-labeled cells was measured by Image J software.</p

Apocynin or urantide inhibited UII-elevated ROS production.

<p>(A) Flow cytometric histogram of DCF in WB-F344 cells. After starvation, cells were treated with UII (10⁻⁹ M) for 2 h, with or without apocynin (0.5 mM) or urantide (1 mM) pre-treatment for 30 min. The relative DCF fluorescence intensity was determined by flow cytometry. Data are presented as mean ± SEM (n = 6). * <i>P</i><0.05 versus control. # <i>P</i><0.05 versus UII group. (B) ROS signals were captured by fluorescent microscopy.</p