Serum miRNA analysis.

<p>Sensitivity and specificity to distinguish between patients with primary sclerosing cholangitis (PSC) and cholangiocarcinoma (CC) for the differentially expressed miRNAs. The analysis was optimized regarding specificity. Table 1 shows sensitivity, specificity, cut off values, area under the curve (AUC) values with confidence interval (CI) and p-values.</p

MiRNA analysis in bile.

<p>Bile validation analysis for patients with primary sclerosing cholangitis (PSC) (n = 52), cholangiocarcinoma (CC) complicating PSC (PSC/CC) (n = 12) and CC (n = 19) revealed significant differences for the different miRNAs. MiR-1537 (<b>A</b>), miR-412 (<b>B</b>), miR-640 (<b>C</b>) and miR-3189 (<b>D</b>) were differentially expressed in patients with PSC and PSC/CC.</p

MiRNA analysis in serum.

<p>Serum validation analysis for patients with primary sclerosing cholangitis (PSC) (n = 40) and cholangiocarcinoma (CC) (n = 31) revealed differentially expressed miRNAs. Fig 2 shows the result for miR-126.</p

Clinical features, demographics and laboratory values of the patient cohorts.

<p>The aforementioned parameters are presented in Table 3 (Bile). Patients with primary sclerosing cholangitis (PSC) and cholangiocarcinoma (CC) were compared regarding demographics, laboratory and microRNA (miRNA) values. Data were expressed as number or median with interquartile range (IQR). ECC: extrahepatic cholangiocarcinoma; ICC: intrahepatic cholangiocarcinoma; ALT: alanine aminotransferase; AST: aspartate aminotransferase; AP: alkaline phosphatase; GGT: gamma-glutamyl transferase; CRP: C-reactive protein; WBC: white blood cells; CA 19–9: carbohydrate antigen 19–9.</p

ROC curves for serum miRNAs.

<p>Receiver operating characteristics (ROC) curves for the detected serum MIRNAs. MiR-1281 and miR-126 showed the highest area under the curve (AUC) value in ROC curve analysis.</p

Bile miRNA analysis.

<p>Sensitivity and specificity to distinguish between patients with primary sclerosing cholangitis (PSC) and cholangiocarcinoma complicating PSC (PSC/CC) in bile for the differentially expressed miRNAs. The analysis was optimized regarding specificity. Table 4 shows the sensitivity, specificity, cut off values, area under the curve (AUC) values with confidence interval (CI) and p-values.</p

Aromatase upregulation by desflurane <i>in vivo</i> and <i>in vitro</i>.

<p>Male mice were treated with 1 MAC desflurane for 15 minutes, then were euthanized 15 minutes later (DES15) or 48 hours later (DES48). The hearts from untreated animals served as native control (Native). Total RNA was extracted from myocardial tissue and aromatase gene (Cyp19a1) expression was analyzed by quantitative real- time PCR (qRT-PCR), n = 5/group (A). Aromatase protein expression <i>in vitro</i> was studied by western blotting. Endothelial cells were treated with oxygen: desflurane (1 MAC) mixture for 15 minutes and collected 15 minutes later (DES0.25), 24 hours later (DES24) or 48 hours later (DES 48). Cells treated with oxygen alone for 15 minutes and collected 15 minutes later, 24 hours later and 48 hours later were assigned as controls (CON0.25, CON 24 and CON 48, respectively). The cells kept under standard conditions served as native control (B). The chart in panel B represents statistical analysis of aromatase signal quantification (n = 4). Aromatase signal in CON group was set to 1 (gray bars) and desflurane influence on aromatase protein expression was compared to CON group (solid bars). GAPDH protein served as internal loading control. Representative aromatase western blot is shown in lower part of panel B. The data are presented as mean± SEM and the <i>P</i> value <0.05 was considered as statistically significant.</p

Summary charts of area at risk and infarct size measurements in male mice hearts.

<p>After hemodynamic measurements, the animals were perfused with triphenyltetrasolium (TPP) and Evans Blue to assess perfused myocardium, area at risk and infracted (necrotic) tissue. Myocardial infarct size (IS) was expressed as a percentage of area at risk (AAR). Values are mean percent± SEM, n = 6 animals/group. A <i>P</i> value <0.05 was considered as statistically significant.</p

Aromatase expression in endothelial and smooth muscle cells.

<p>Aromatase cellular localisation pattern in endothelial (EC) and smooth muscle cells (SMC) was assessed by immunocytochemistry and visualized using confocal imaging technique. Aromatase was immunostained using specific primary antibody and visualized by addition of the secondary antibody conjugated with AlexaFluor594 fluorophore (red). Aromatase was abundant in the cytosolic fraction of endothelial (A) and smooth muscle cells (C). Cell nuclei were detected using lamin A/C antibody and visualized using AlexaFluor488- labelled secondary antibody (green). Merge images B and D show aromatase and lamin A/C co- immunostaining.</p

Aromatase immunostaining in mouse heart and aorta.

<p>Aromatase protein was abundant in mouse myocardium (B, solid arrows) and in the endothelium (solid arrows) as well as in media (white arrows) in the aorta (D). Aromatase signal was visualized by diaminobenzidine substrate (DAB) combined with hematoxylin/eosin staining. Primary antibody was omitted in negative control staining (A and C).</p