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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. 2. High glucose enhances CIKS expression via increased transcription.

Fig. 2

A, HG induces CIKS mRNA expression. At 70% confluency, the complete medium was replaced with EBM-2 (without supplements) for 2h, and then incubated for the indicated time periods in the presence of 25 mM D-glucose. CIKS mRNA was analyzed by RT-qPCR, and 18S served as an invariant control. *P < at least 0.01 vs. NG (n=6). B, HG induces CIKS protein expression. HAEC treated as in A were analyzed for CIKS protein expression by immunoblotting. Actin served as a loading control (n=3). C, HG induces CIKS expression via increased transcription. HAEC were treated with HG with actinomycin D (ActD) or cycloheximide (CHX) for 30 min, and analyzed for CIKS mRNA expression by RT-qPCR. *P < at least 0.01, †P < 0.01 vs. NG (n=6). D, E, HG stimulates CIKS promoter-dependent reporter gene activation via IRF, c/EBP and AP-1. HAEC were transfected with a reporter vector containing a 200-bp fragment of the 5′-flanking region of the human CIKS gene (3 g for 24 h) with and without mutations (D). pGL3-Basic served as a vector control. Cells were co-transfected with the Renilla luciferase vector (100 ng). Following transfection, cells were treated with HG for 12 h and harvested for the dual-luciferase assay. Firefly luciferase data were normalized to that of corresponding Renilla luciferase activity. E, *P < at least 0.001 vs. NG, †P < at least 0.05 vs. HG, ††P < 0.001 vs. HG (n=12).