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. Author manuscript; available in PMC: 2014 Jan 1.
Published in final edited form as: Cell Signal. 2012 Oct 17;25(1):359–371. doi: 10.1016/j.cellsig.2012.10.009

Fig. 3. High glucose-induced CIKS expression is ROS dependent.

Fig. 3

A, HAEC expresses relatively more Nox4 than Nox1 and Nox2 under basal conditions. DNA-free total RNA from HAEC was analyzed for Nox1, 2 and 4 by RT-PCR (n=3). B, C, HG induces ROS generation. Cells were treated with NAC (5 mM for 1 h), DPI (10 mM in DMSO for 1 h) or infected with Ad.siNox4 (MOI 100 for 24 h) prior to HG addition. Ad.siGFP served as a control. Intracellular ROS levels following HG treatment were determined by oxidation of the cell-permeable, redox-sensitive fluorophore DCFH-DA into fluorescent DCF (B; knockdown of Nox4 by immunoblotting is shown on the right) and by lucigenin-enhanced chemiluminescence (C). C, *P < at least 0.01 vs. NG, †P < 0.01 vs. HG (n=6). D, E, F, HG-induced CIKS expression is dependent on DPI-inhibitable Nox4-dependent ROS generation. HAEC treated with DPI (C), NAC (D), or infected with Ad.siNox4 (E) were treated with HG (25 mM for 2 h), and analyzed for CIKS expression by immunoblotting using whole cell lysates. Densitometric analysis of three independent experiments is summarized on the right (D) or in lower panels (E, F). D, E, F, *P < 0.01 vs. NG, †P < 0.05 vs. HG.