Suppression of NQO1 protein expression by cortisone in 11β-HSD1 expressing H4IIE cells but not in pCDNA3 transfected cells.

<p>H4IIE cells transiently transfected with either pCDNA3 or 11β-HSD1 were treated for 24 h with vehicle (DMSO), cortisone, sulforaphane or cortisone and sulforaphane (<i>upper panel</i>). Cells were lysed, and equal protein amounts were used for Western blot analysis. Samples were probed for NQO1 using actin as a loading control. <i>Lower panel</i>, densitometric analysis of NQO1 bands normalized against b-actin. Graphs are representative of three independent experiments.</p

Suppression of Nrf2-dependent reporter gene activation by glucocorticoids in HEK-293 cells.

<p>HEK-293 cells transiently transfected with plasmids for Nrf2, GR, ARE8L-reporter, pCMV-LacZ and either pcDNA3 (<i>A, B</i>) or 11β-HSD1 (<i>C</i>) were incubated for 24 h with vehicle (DMSO), 10 µM sulforaphane, 100 nM cortisone or cortisol, in the presence or absence of 1 µM T0504 or RU-486. Data (mean ± SD) were obtained from three independent experiments each measured in triplicate. *, <i>p</i><0.05, **, <i>p</i><0.01, ***, <i>p</i><0.001, <i>p</i>-value was obtained using one-way ANOVA followed by Bonferroni post-tests compared with vehicle control (DMSO).</p

11β-HSD1-mediated suppression of Nrf2-induced NQO1 expression in H4H1 cells.

<p>H4H1 cells were incubated for 24 h at 37°C with 10 µM sulforaphane in the absence or presence of 100 nM cortisone and 1 µM glycyrrhetinic acid (GA), followed by quantification of mRNA levels by real-time RT-PCR. Data (mean ± S.D. from three independent experiments performed in triplicate) represent ratios of NQO1 mRNA to GAPDH control mRNA from treated cells normalized to the values obtained from cells incubated vehicle (DMSO). *, <i>p</i><0.05, **, <i>p</i><0.01, ***, <i>p</i><0.001, <i>p</i>-values were obtained using one-way ANOVA followed by Bonferroni post-tests compared with vehicle control (DMSO).</p

Increased susceptibility of 11β-HSD1 expressing cells to H₂O₂-induced oxidative stress.

<p>H4IIE cells transiently transfected with pCDNA3 (<i>A</i>), pCDNA3 or 11β-HSD1 (<i>B, C</i> and <i>D</i>) were treated for 24 h with vehicle, 100 nM cortisol (<i>A</i>), 100 nM cortisone (<i>C</i>) or simultaneously with cortisone and 1 µM T0504 (<i>B, D</i>). The medium was replaced by assay buffer (HBSS) containing 1 g/L glucose. Single cell real-time measurements were performed on a Leica SP5 confocal microscope. After 5 min baseline adaption, cells were exposed to a final concentration of 10 µM (<i>A, B</i>) or 100 µM (<i>C, D</i>) H<b>₂</b>O<b>₂</b> and responses were compared between differentially transfected or vehicle treated cells over a period of 30 min (<i>A, B</i>) or 45 min (<i>C</i>). Data represent mean ± SEM of seven different cells for each transfection. *, <i>p</i><0.05, **, <i>p</i><0.01, ***, p<0.001, <b><i>p</i></b>-value was obtained using one-way ANOVA followed by Bonferroni post-tests compared with pcDNA3 transfected cells. To analyze the total cell population (<i>D</i>), H4IIE cells 4,000,000 cells/mL were resuspended in assay buffer. Suspensions of cells (100 µL) treated either with cortisone or cortisone and T0504 were transferred into a 96-well plate, centrifuged for 2 min at 180× g and challenged by adding 100 µL assay buffer containing 100 µM H<b>₂</b>O<b>₂</b>. Fluorescence was immediately measured after adding H₂O₂ and data were collected every 27 s at 37°C for 9 h. One of three representative experiments is shown (<i>D</i>).</p

Rat Genome 230 2.0 Affymetrix chip analysis.

<p>RNA purified from whole liver tissues of ten male and ten female rats was hybridized to Rat Genome 230 2.0 Affymetrix chips. Gender-specific differences in the expression of the HSD11B1, HMOX1, NQO1 and ABCC3 genes were assessed. The data represent fold change in gene expression (male vs. female). The statistical relevance was assessed by multiple unpaired t-tests, with Benjamini Hochberg FDR multiple testing correction, <i>p</i>≤0.01.</p

Suppression of Nrf2 and NQO1 protein expression by cortisol in oxidative stress-induced H4IIE cells.

<p>H4IIE cells were treated for 24 h with vehicle (DMSO), cortisone, sulforaphane or cortisone and sulforaphane in the presence or absence of H₂O₂. Cells were lysed, and equal protein amounts were used for Western blot analysis. Samples were probed for Nrf2 and NQO1 using actin as a loading control. The <i>lower panel</i> shows a densitometric analysis of Nrf2 (<i>left</i>) and NQO1 (<i>right</i>) protein normalized against β-actin. A representative experiment is shown.</p

Inhibition of Nrf2-induced mRNA expression of NQO1 and GSTA2 by cortisol.

<p>H4IIE cells were incubated for 24 h at 37°C with 10 µM sulforaphane in the absence or presence of 100 nM cortisol or cortisone, respectively, followed by determination of NQO1 (A) and GSTA2 mRNA levels (B) by real-time RT-PCR. Data (mean ± S.D. from three independent experiments performed in triplicate) represent ratios of NQO1 and GSTA2 mRNA to GAPDH control mRNA from treated cells normalized to the values obtained from cells incubated with vehicle (DMSO). *, <i>p</i><0.05, **, <i>p</i><0.01, ***, <i>p</i><0.001, <i>p</i>-values were obtained using one-way ANOVA followed by Bonferroni post-tests compared with vehicle control (DMSO).</p

Heart Structure-Specific Transcriptomic Atlas Reveals Conserved microRNA-mRNA Interactions

<div><p>MicroRNAs are short non-coding RNAs that regulate gene expression at the post-transcriptional level and play key roles in heart development and cardiovascular diseases. Here, we have characterized the expression and distribution of microRNAs across eight cardiac structures (left and right ventricles, apex, papillary muscle, septum, left and right atrium and valves) in rat, Beagle dog and cynomolgus monkey using microRNA sequencing. Conserved microRNA signatures enriched in specific heart structures across these species were identified for cardiac valve (miR-let-7c, miR-125b, miR-127, miR-199a-3p, miR-204, miR-320, miR-99b, miR-328 and miR-744) and myocardium (miR-1, miR-133b, miR-133a, miR-208b, miR-30e, miR-499-5p, miR-30e*). The relative abundance of myocardium-enriched (miR-1) and valve-enriched (miR-125b-5p and miR-204) microRNAs was confirmed using in situ hybridization. MicroRNA-mRNA interactions potentially relevant for cardiac functions were explored using anti-correlation expression analysis and microRNA target prediction algorithms. Interactions between miR-1/Timp3, miR-125b/Rbm24, miR-204/Tgfbr2 and miR-208b/Csnk2a2 were identified and experimentally investigated in human pulmonary smooth muscle cells and luciferase reporter assays. In conclusion, we have generated a high-resolution heart structure-specific mRNA/microRNA expression atlas for three mammalian species that provides a novel resource for investigating novel microRNA regulatory circuits involved in cardiac molecular physiopathology.</p> </div

Anti-correlated microRNA targets are directly inhibited by microRNA over expression.

<p>(A–D) Real-Time RT-PCR of Timp3, Rbm24, Tgfbr2 and Csnk2a2 in HPASM cells transfected with mimics for miR-1, miR-125b-5p, miR-204, miR-499 and miR-208b or with a mimic microRNA negative control. Data were normalized to 18S RNA. (E-H) Luciferase activity of wild-type (WT) or mutant (MUT) Timp3, Rbm24, Tgfbr2 and Csnk2a2 3′-UTR reporter genes cotransfected with their paired microRNA mimics. Data were averaged for n = 4 in 3 independent experiments and error bars represented standard deviation of the mean. P values: * = P<0.05, ** = P<0.01, *** = P<0.005.</p

Cross-species microRNA/mRNA cardiac atlas.

<p>Data sets obtained for rat, dog and cynomolgus monkey are indicated. Inset: magnified view of a papillary muscle.</p