Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Detection of PAI-1 mRNA by qRT-PCR in various cell lines.

(1)<p>Cycle Threshold (Ct) values.</p>(2)<p>Relative transcript expression levels were calculated by use of the the 2^−ΔΔCT method using HUVEC as reference.</p

Influence of miR-421 and miR-30c binding to total 3′UTR <i>SERPINE1</i> on luciferase activity.

<p><b>A.</b> Psicheck2 vector containing total 3′UTR <i>SERPINE1</i> sequence fused to renilla luciferase was co-transfected with Pre-Neg, Pre-miR-30c, Pre-miR-421 or both Pre-miR-30c and Pre-miR-421. Graph shows renilla luciferase activity normalized to firefly luciferase activity and expressed as percentage of Pre-Neg transfected cells (n = 6, p<0.005,***). <b>B.</b> Psicheck2 vector containing total 3′UTR <i>SERPINE1</i> sequence with the mutated rs1050955-A allele fused to renilla luciferase was co-transfected with Pre-Neg, Pre-miR-421, Pre-miR-30c or both. Graph shows renilla luciferase activity normalized to firefly luciferase activity and expressed as percentage of Pre-Neg transfected cells (n = 5 ** p<0.01). <b>C.</b> Comparison of miR-421 inhibitory effect on luciferase activity between <i>SERPINE1</i> 3′UTR wild-type or mutated at rs1050955 (n = 4).</p

Influence on luciferase activity of miR-421 and miR-30c binding to the 3′UTR <i>SERPINE1</i> 1704–1760 region.

<p><b>A.</b> psicheck2 vector containing 3′UTR <i>SERPINE1</i> sequence surrounding miR-30c predicted binding site or miR-421 predicted binding sites according to the allele present at rs1050955 fused to renilla luciferase were co-transfected with Pre-Neg, Pre-miR-30c or Pre-miR-421. Graph shows renilla luciferase activity normalized to firefly luciferase activity and expressed as percentage of Pre-Neg transfected cells (n = 4 for miR-30c and n = 5 for miR-421; *, p<0.05; **, p<0.01)). <b>B.</b> Various 3′UTR <i>SERPINE1</i> sequence containing both miR-421 site 1 and site 2 predicted binding sites or mutation of each seed sequence binding sites were fused to renilla luciferase. Plasmids were transfected with Pre-Neg or Pre-miR-421. Graphs show renilla luciferase activity normalized to firefly luciferase activity and expressed as percentage of Pre-Neg transfected cells (n = 5 except for site1+2 1 mut 2 mut construct, n = 4; *, p<0.05; **, p<0.01).</p

Plasma levels of miR-421 and miR-30c in plasma samples of venous thrombosis patients.

<p>MiR-421 and miR-30c were detected by qRT-PCR in plasma samples from two groups of 20 patients either with low (1.6+/−1 ui/ml) or high (40.5+/−13 ui/ml) PAI-1 plasma levels. 40 fmol of synthetic cel-miR (39/54/238) were used for normalization. Median values are shown in black line (*: p<0.05).</p

Detection of miRNAs of interest by qRT-PCR in various cell lines.

(1)<p>MiRNAs with Cycle Threshold (Ct) values over 31 were considered not sufficiently expressed for further investigations.</p>(2)<p>Relative transcript expression levels were calculated by use of the the 2^−ΔΔCT method using HUVEC as reference.</p

MiR-421 and miR-30c inhibit <i>SERPINE1</i> expression in HUVEC.

<p><b>A.</b> Quantification by qRT-PCR of PAI-1 mRNA level after over-expression of either Pre-miR-421, Pre-miR-30c or a Pre-miR Negative control (Pre-Neg) in HUVEC cells. RPL32 mRNA level was used for normalization and data shown were expressed as percentage compared to Negative control (*, p<0.05 n = 4). <b>B.</b> Western-Blot and quantification of PAI-1 and GAPDH protein level after over-expression of either Pre-miR-421, Pre-miR-30c or both compared to Pre- Neg transfected cells. Data shown were normalized to GAPDH protein level and expressed as percentage compared to Negative control (n = 5 for miR-421 and miR-30c, n = 3 for miR-421+30c; *, p<0.05; **, p<0.01).</p

miRNA binding sequences in the <i>SERPINE1</i> 3′UTR region.

<p>The 3′UTR human <i>SERPINE1</i> sequence is 1841 bp long. The rs1050955-A muted allele is shown in red bold. <b>A. </b><i>SERPINE1</i> DNA sequence showing the rs1050955 G/A polymorphism in position 1737. <b>B.</b> Representation of various miRNA seed region and their complementary sequence in <i>SERPINE1</i> 3′UTR 1728–1756 region. <b>C.</b> Alignment of miR-421 onto <i>SERPINE1</i> 3′UTR 1704–1760 region which includes two predicted binding sites, site 1 and site 2, complementary to the miR-421 seed sequence.</p

Maintenance of biological variation after quantile normalization and <i>ComBat</i>.

<p>To assess whether biological sources of variability were maintained after batch effect removal, associations between each probe and body mass index (BMI) were calculated using linear mixed models within each batch containing 1092 samples before and after applying <i>ComBat</i>. A, B: For each probe, we plotted the effect of BMI on expression in ComBat corrected data on the x-axis and the quantile-normalized but uncorrected on the y-axis. BMI beta estimates were highly correlated between corrected and uncorrected datasets in A: BL samples (R = 0.995) and B: FU samples (R = 0.986). C, D: The effect of BMI on gene expression in BL samples was plotted against the effect observed in FU samples after C: quantile normalization and D: quantile normalization followed by <i>ComBat</i>. The correlation between BMI effect estimates at BL and FU was slightly higher for <i>ComBat</i> corrected data (r = 0.787) compared data, which was only quantile normalized (r = 0.781).</p

Comparison of different batch effect removal approaches.

<p>Replicates of RNA samples extracted at BL were hybridized on Illumina HT12 microarrays at both examination dates. Overall gene expression rescaled by seven different approaches. Components of variance are visualized as PCA plots. Replicate samples extracted and measured at baseline (BL_rep) are marked red and repeated measures at 5-year follow up (BLFU_rep) in blue. Correction based on A: Deming regression, B: Passing-Bablok regression, C: linear mixed models, D: 3^rd order polynomial regression and E: <i>qspline</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156594#pone.0156594.ref010" target="_blank">10</a>] was not capable to remove batch effects from gene expression data. The PCA plots show clusters between replicates extracted, processed and hybridized at both time points. F: After applying <i>ComBat</i>, G: quantile normalization followed by <i>ComBat</i>, H: <i>ReplicateRUV</i> and I: quantile normalization plus <i>ReplicateRUV</i>, no clustering of samples was observed indicating successful removal of batch effects.</p