Immunohistochemical staining and analysis of NF-κB activation in TNF-α and everolimus treated endothelial cells.

<p>HCAEC cells were treated for 1 hour with MesoEndo Medium (Baseline), 100 ng/ml TNF-α with or without 0.5 μM everolimus or cytokine with the solvent of everolimus (TNF-α+DMSO). Nuclear localization of the NF-κB p65 subunit was monitored by immunostaining. Green: p65 staining; blue: cell nuclei. Scale bar: 20 μm (A). Ratio of the fluorescence intensity of the NF-κB immunostaining in cell nuclei and cytosol was analyzed (B). Mean ± SEM, n = 6-8/group.</p

Quantification of circulating miR-155, miR-185 and miR-181b by RT-qPCR in plasma samples of BMS and DES patients.

<p>After total RNA isolation, the expression of circulating miRNAs was quantified in plasma samples by UPL-probe based stem-loop RT-qPCR assay. Plasma miR-155 (A) and miR-185 (B) were significantly upregulated in BMS patients with ISR compared to BMS and DES subjects without any complications, while miR-181b levels (C) were lower in those with ISR versus others without complication. (All DES: n = 21, all BMS: n = 28, ISR: n = 6).</p

Analysis of E-selectin and VCAM-1 expression at mRNA and protein levels in HCAECs after TNF-α stimulation.

<p>HCAEC cells were treated with recombinant TNF-α (100 ng/mL) for 1–24 hours to generate cellular inflammatory conditions. Elevated E-selectin (SELE) (A) and VCAM-1 mRNA levels (B) induced by TNF-α already after 1 hour were downregulated by everolimus in HCAECs <i>in vitro</i>. In parallel, soluble E-selectin (C) and VCAM-1 concentrations (D) were measured by ELISA in the supernatants of ECs and were also significantly decreased after 4 and 24 hours, respectively. Mean ± SEM, n = 4-8/group.</p

Overexpression of miR-181b altered the levels of SELE and VCAM1 mRNA in HCAECs.

<p>The direct association between miR-181b and SELE/VCAM1 was investigated in HCAECs after stimulation with TNF-α by using transfection of mirVana^® miR-181b mimics (25 pmol) with Lipofectamine RNAiMAX^® Transfection Reagent for 24 hours. In parallel, negative control samples were treated with mirVana^® miRNA mimic negative control (NEG-01, 25 pmol). After transfection, miR-181b with SELE and VCAM1 mRNAs were quantified by RT-qPCR. Highly increased miR-181b levels (A) resulted in significantly lowered SELE and VCAM1 mRNAs compared to NEG-01 control samples (100%, B). **P = 0.006, ***P<0.001 based on t-test. Mean ± SEM, n = 4/group.</p

Sequences of matured small RNA molecules investigated. All sequences are in written in 5′-3′ direction.

<p>Sequences of matured small RNA molecules investigated. All sequences are in written in 5′-3′ direction.</p

Schematic figure about the model to demonstrate the regulatory mechanisms of everolimus on E-selectin (SELE) and VCAM-1 expression.

<p>Everolimus decreases EC activation via suppressing the NF-κB pathway with decreased p65 translocation into cell nuclei causing the modulation of E-selectin and VCAM-1 expression as well as miR-181b level at transcriptional and post-transcriptional level, respectively. EC: endothelial cell, TNF-α: tumor necrosis factor alpha, SELE: E-selectin, VCAM-1: vascular cell adhesion molecule-1, RISC: RNA-induced silencing complex.</p

Sensitivity and specificity of miRNA specific UPL-based quantitative PCR system.

<p>Amplification plot of mmu-mir-1 in range from 10 ng to 10^–3 ng input mouse heart total RNA (A). Sequence similarities and differences between mir-181a, b, and c (B). Amplification plot of synthetic mir-181a miRNA ranging from 10 pM to 10^–4 pM input mir-181a amplicon (C). Standard curve of synthetic mir-181a miRNA (D). Specificity and relative detection capacity of mir-181 specific UPL-based qPCR assays. Numbers represent the percentage of the signals measured on the synthetic amplicons. 100% is always the signal measured by an assay on its specific synthetic amplicon, like mir-180a assay on the mir-181a synthetic amplicon. In brackets the corresponding Cp values are shown.(E).</p

Schematic description of small RNA specific UPL-based quantitative PCR assay and our oligo design system.

<p>Two steps small RNA specific UPL-based quantitative PCR assay relies on reverse transcription using small RNA specific stem-loop RT primer and real-time quantitative PCR reaction using small RNA specific forward primer, UPL21 probe and universal reverse primer (A). Workflow of our oligo design system (B). Primers and probe for the designed hsa-mir-181a specific UPL-based quantitative PCR assay (C).</p

Measurement of siRNA expression with small RNA specific UPL-based quantitative PCR assays.

<p>Sequence specificity of PRMT1 specific siRNA for exon 3 of PRMT1 (A). PRMT1 specific siRNA levels as detected by qPCR and the corresponding mPRMT1 mRNA as well as protein levels detected by qPCR and Western blot analysis (B).</p

Sequences of oligonucleotides used for qPCR measurements. UPL probe number 21 was used for the measurements.

<p>Universal reverse primer in all cases was: GTGCAGGGTCCGAGGT. All sequences are in written in 5′-3′ direction.</p