Effect of insulin on the doxorubicin-induced changes in Sp1 and p53.

<p>(A, D) H9c2 cardiac myocytes were left untreated or treated with the indicated concentration of doxorubicin (<i>Doxo</i>) for 12 h, and (B, C, E) serum-deprived cells were left untreated or pretreated with insulin (200 nM) for 1 h prior to treatment with doxorubicin (1 μM) for 12 h. Whole cell lysates were analyzed by immunoblotting with anti-Sp1, anti-phospho-p53 (Ser¹⁵), anti-p53 and anti-GAPDH antibodies. Graphs represent the mean ±S.D. of the normalized densitometric analyses of Sp1 protein levels (A, **<i>p</i> < 0.01, n = 3; B, ***<i>p</i> < 0.001, n = 5). (D, E) Sp1 mRNA amount was determined by RT-PCR (28 cycles). Note that the blots in <i>panel C</i> represent one of three independent experiments.</p

Inhibitory effect of insulin on the doxorubicin-induced Sp1 degradation via PI3K/Akt/mTORC1 pathway in H9c2 cardiac myocytes.

<p>(A) Serum-deprived H9c2 cardiac myocytes were left untreated or pretreated with 200 nM insulin (<i>Ins</i>) for 1 h and treated with 1 μM doxorubicin (<i>Doxo</i>) for 12 h. (B–D) Cells were pretreated with 2 μM PI3K inhibitor LY294002 (<i>LY</i>) (B), 1 μM mTORC1 inhibitor rapamycin (<i>Rapa</i>) (C), or 5 μM p70S6K inhibitor PF4708671 (<i>PF</i>). (D) for 1 h, followed by treatment with insulin (200 nM) for 1 h and then with doxorubicin (1 μM) for 12 h. Whole cell lysates were analyzed by immunoblotting for protein levels or phosphorylation status of Akt, mTORC1 and p70S6K and for protein levels of Sp1 using antibodies listed in Materials and Methods. Note that these blots represent one of three independent experiments. Graphs represent the mean ±S.D. of the normalized densitometric analyses of Sp1 protein levels (*<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001, n = 3).</p

Effect of doxorubicin and/or insulin on the transcriptional activity of Sp1 and p53.

<p>(A) Multiple sequence alignment (ClustalW2) of the proximal promoter regions of the rat, mouse and human <i>survivin</i> genes. Black arrow indicates the transcriptional start site and +1 points out the translation start codon. Canonical p53, Sp1, Sp1-like sites of the mouse and human <i>survivin</i> genes are boxed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref012" target="_blank">12</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref038" target="_blank">38</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref040" target="_blank">40</a>]. Two gray bars indicate the primers for ChIP analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref031" target="_blank">31</a>]; a red bar corresponds to human CHR sequences; two blue bars highlight human CDE regions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref050" target="_blank">50</a>]. (B) Serum-deprived cells were left untreated or pretreated with 200 nM insulin (<i>Ins</i>) for 1 h and treated with 1 μM doxorubicin (<i>Doxo</i>) for 12 h. Cross-linked cell lysates were subjected to ChIP analysis with anti-Sp1 or anti p53-antibody. RT-PCR (36 cycles) was performed with ChIP primers as listed in Materials and Methods (n = 5). (C–E) One day after transfection with Sp1 siRNA (20 nM), cells were left untreated or pretreated with insulin (200 nM) for 1 h and treated with doxorubicin (1 μM) for 24 h. Whole cell lysates were immunoblotted with anti-Sp1, anti-survivin and anti-caspase-3 (active form) antibodies (C, D), and total RNA was analyzed by RT-PCR (28 cycles) with specific primers to <i>survivin</i> gene (E). Note that these results represent one of three independent experiments.</p

Effects of sRAGE on mRNA Levels in Aorta of AngII-induced Apo E KO mice.

<p>A. Reverse transcription PCR analysis of RAGE, ICAM-1 and MCP-1 gene expression. Data in histograms represent mean ± SD from 3 experiments. Lane 1, saline injection; lane 2, infusion of AngII; lane 3, infusion of AngII with 0.5 µg/day of sRAGE; lane 4, infusion of AngII with 1 µg/day of sRAGE; lane 5, infusion of AngII with 2 µg/day of sRAGE. B. RAGE, ICAM-1 and MCP-1 mRNA expression level were determined by Real-Time PCR. Results are means ± SD from 3 experiments each performed in duplicate. *<i>p</i><0.05, **<i>p</i><0.01, *** <i>p</i><0.001 and ns = non-significant.</p

Quantification of Atherosclerosis.

<p>A. Photomicrographs of atherosclerotic lesions from aortic sinuses of Apo E KO saline injection mice, AngII infused, and AngII infused with various doses of sRAGE (n = 7); scale bar, 200 µm. B. Quantitative data, *<i>p</i><0.01.</p