Quantitative PCR primers.

<p>Quantitative PCR primers.</p

Effect of adenosine on lymphatic vasculature and inflammatory cell recruitment in the liver.

<p>Control adenovirus expressing GFP (CTRL virus) or adenovirus carrying the sequence of 5-nucleotidase (cN-IA virus) were injected in the caudal vein of mice. PBS was used as negative control (PBS). Mice were sacrificed 2 weeks after injection. The efficacy of virus transduction was shown by GFP immunostaining (a) and by cN-IA immunostaining (b)). Liver sections were stained with anti-podoplanin antibodies to detect lymphatic vessels (c), with anti-CD45 antibodies to detect inflammatory cells (d), and with anti-F4/80 antibodies to detect macrophages (e). Data are presented as mean ± SEM (n = 15 for PBS, n = 13 for GFP and n = 12 for cN-IA).**p<0.01, *** p<0.001. Representative pictures are shown.</p

Effect of adenosine on LEC tube formation.

<p>Tube formation was assessed in two in vitro models, the tubulogenesis assay (a) and the spheroid assay (b). HMVEC-dLy were cultured in 2% FBS medium (CTRL) and treated or not with EHNA alone (EHNA 10 μM) or EHNA with different concentrations of adenosine (AE). Quantification of tube formation (a) and cell migration (b) was performed by a computerized method on pictures taken after 24 h of culture. The parameters measured are: the tubes branching (branching), the length of tube (length), the surface occupied by tube (surface), and the maximal length of tube (Lmax). * p<0.05 vs CTRL, **p<0.01 vs CTRL, *** p<0.001 vs CTRL. Each experiment was performed three times and representative pictures are shown. Data are expressed as mean ± SEM.</p

Effect of adenosine on LEC migration in scratch test.

<p>HMVEC-dLy were treated during 24 h in medium containing 2% FBS, in the presence of EHNA alone (EHNA, 10 μM) or EHNA with different concentrations of adenosine (AE). Pictures were taken at 0 h, 8 h, 16 h and 24 h after insert removing. Results are expressed as percentage of wound closure (percentage closure) (mean ± SEM). * p<0.05 vs CTRL, ***p<0.001 vs CTRL. Each experiment was performed three times and a representative picture of each condition is shown.</p

Effect of adenosine on LEC viability and proliferation.

<p>Cell viability (a) and proliferation (b–c) were evaluated in HMVEC-dLy ( = LEC) cultured for 48 h (left panel) or 72 h (right panel) in medium containing 2% FBS (CTRL). Cells were treated with EHNA alone (EHNA 10 μM), EHNA with different concentrations of adenosine (AE), the A2a agonist CGS21680 or the A2b agonist NECA. For cell viability (a), results are expressed as percentage of dead cells (black) and living cells (white). Cell proliferation (b–c) was measured with a CyQANT assay. * p<0.05 vs control (CTRL), *** p<0.001 vs CTRL. Data are expressed as mean ± SEM (n = 3).</p

Effects of adenosine on lymphangiogenesis in the in vivo model of collagen sponge.

<p>Sponges were soaked with PBS as control (CTRL), with VEGF-C (1 ng/ml) as positive control (VEGF-C), with 20 μl CADO (3 ng/ml), a stable analog of adenosine, or with CADO in presence of the A2b antagonist MRS1754 (2.4 mg/ml). Sponges were implanted between the two skin's layers of ear's mice for 3 weeks. Every other day, PBS or MRS1754 were injected in the apex of the ear. Sponge sections were stained with an anti-Lyve-1 antibody to detect lymphatic vessels (green) and Dapi to detect cell nucleus (blue). The graph corresponds to computerized quantification of the surface occupied by lymphatics (vessel area). Data are expressed as mean ± SEM (n = 6). ** p<0.01 vs CTRL.</p

Effect of adenosine on the migration of LEC in Boyden chamber.

<p>HMVEC-dLy were cultured in medium containing 2% FBS (control condition, CTRL), with or without 10 μM EHNA and 0.1–10 μM adenosine (AE). Cell migration was assessed in a Boyden chamber assay 24 hours after treatment onset. There was no difference on cell migration between all treatments. Results are expressed as percentage of migrating cells (mean ± SEM, n = 3).</p

Added value of combinations of miRNAs (logistic regression).

<p>Shown are the results of all combinations of miRNAs added to model 1. The Wald chi square test indicates the overall significance of the model. The likelihood ratio test (LRT) compares the fit of a model with miRNAs to model 1. AIC: Akaike information criteria.</p

Added value of combinations of miRNAs (censored regression).

<p>Shown are the results of all combinations of miRNAs added to model 3. The Wald chi square test indicates the overall significance of the model. The likelihood ratio test (LRT) compares the fit of a model with miRNAs to model 3. AIC: Akaike information criteria.</p

Bootstrap internal validation (censored regression).

<p>Represented is the percentage of times a combination of miRNAs was selected as providing the best improvement of the prediction of model 3 over 150 bootstrap iterations.</p