A cleaved form of vimentin was more abundant in RHD valve samples.

<p><b>(A)</b> Immunoblots showing vimentin expression in valve samples of RHD (N = 8), MXD (N = 8) and controls (CTL, N = 6). (<b>B)</b> and <b>(C)</b> Densitometry analysis of the relative expression between band 2 (45 kDa), or band 3 (42 kDa), and 1 (54 kDa). (<b>D)</b> Complete sequence of vimentin. Highlighted sequences (boxes) represent peptide sequences of RHD valve spots of 2D-gel analysis previously identified by mass spectrometry (LC-ESI-MS/MS)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170191#pone.0170191.ref007" target="_blank">7</a>]. Statistical analysis was performed using the ANOVA (Kruskal Wallis) test. <i>p</i>-value< 0.05 was considered significant.</p

Co-localized expression of collagen VI and lumican in MXD valvular tissue and RHD stenotic valve analyzed by confocal microscopy.

<p>(<b>A</b>) Control valve tissue from cadaveric transplantation donor; (<b>B</b>) Valve tissue from MXD patient (#15); (<b>C</b>) Valve tissue from RHD patient with stenosis (# 6). Red- collagen type VI; green- lumican; blue—DAPI.</p

Mitral valve immunohistological pattern expression of vimentin, collagen VI and lumican in damaged valves of RHD and MXD patients.

<p>Mitral valve samples of one cadaveric donor (<b>A)</b> and MXD (<b>F</b>) and RHD (<b>K</b>) patients were stained by hematoxylin-eosin (HE). Immunoperoxidase reactions performed for cadaveric donor, MXD patient (#15) and RHD patients with regurgitation (# 6) and stenosis (#5) for vimentin (<b>B</b>, <b>C</b>, <b>D</b> and <b>E</b>); collagen VI (<b>G</b>, <b>H</b>, <b>I</b>, <b>J</b>) and lumican (<b>L</b>, <b>M</b>, <b>N</b>, <b>O</b>).</p

Relative expression of N-terminal and C-terminal portions of vimentin in valves of RHD and MXD patients.

<p>Immunoblots of valve proteins in SDS-PAGE gels were made with antisera raised against vimentin N-terminal or C-terminal peptides. Relative protein expression levels were measured by densitometry. RHD valve samples (n = 8), MXD valve samples (n = 8) and controls (n = 6). Vimentin N-term and C-term: N and C terminal fragments; N/C ratio, ratio between relative protein levels of vimentin N-term and C-term fragments. Statistical analyses were performed using the ANOVA (Kruskal-Wallis) test.</p

Clinical data and mitral histological findings.

<p>Clinical data and mitral histological findings.</p

Relative expression of collagen-VI, lumican and vitronectin in the valve tissue of RHD patients.

<p><b>(A)</b> Immunoblots showing collagen-VI, lumican and vitronectin expression in RHD (n = 8), MXD (n = 8) and controls (CTL, n = 6) valve samples. (<b>B, C, D)</b> Densitometry analyses of collagen-VI alpha-chain, vitronectin and lumican were determined. RHD valve tissue (n = 8), MXD (n = 8) and controls (n = 6). Statistical analysis was performed using the ANOVA (Kruskal-Wallis) test. <i>p-</i>value< 0.05 was considered significant.</p

Selected sequences in the N- and C-terminal regions of vimentin for eliciting anti-vimentin rabbit polyclonal sera.

<p>Mass spectrometry-identified peptides from the N- and C-terminal fragments of vimentin were used to induce rabbit polyclonal antisera which were used in subsequent experiments. Antisera were raised by AB-mart Inc. Epitope score plot was performed with a proprietary algorithm from AB-Mart Inc.</p

Effect of serum from DENV-positive patients on TEER of endothelial cells.

<p>Confluent monolayers of HUVECs cultured in ECIS arrays were treated or not with 10% serum from three different groups: Healthy blood donors (control, n = 13), DENV infected patients without leakage (no leakage, n = 13), or DENV infected patients with leakage (leakage, n = 13). (*) Asterisks indicate statistically significant differences (ANOVA/ Tukey’s test) between groups with <i>p</i><0.05. TEER values were obtained at 30 (A) and 120 (B) min after treatment.</p

Serum albumin levels.

<p>Albumin levels of serum from the three different groups: Healthy blood donors (control, n = 13), DENV infected patients without leakage (no leakage, n = 13), or DENV infected patients with leakage (leakage, n = 13) were quantified by the bromocresol purple method. Groups were compared by the ANOVA/Tukey’s test. *Statistically significant difference (<i>p</i><0.05).</p

Real-time impedance measurement throughout 5 h after treatment.

<p>Confluent monolayers of HUVECs cultured in ECIS arrays were treated or not with 10% serum from three different groups: Healthy blood donors (control, n = 13), DENV infected patients without leakage (no leakage, n = 13), or DENV infected patients with leakage (leakage, n = 13). Graph A shows results from the first experiment and graph B from the second experiment.</p