MiR-144 targets 3′UTR of CFTR.

<p>Cells were transfected with 50 ng of psiCHECK containing WT or Mut CFTR 3′UTR and either 30 or 60 nM of pre-miR-144. Twenty four hours following transfection, cells were assayed for both firefly and renilla luciferase using the dual luciferase glow assay. All transfection experiments were conducted in triplicate. Data are expressed as mean±SE of at least three independent experiments. *p<0.05, n.s.: not significant.</p

Histopathological and ultrastructural analysis of the heart.

<p>(a) Heart of a non-dengue case stained with H.E. and presenting normal aspect. (b and c) Heart sections of dengue cases, stained with HE, showing cardiac injuries, including hemorrhage (He), edema (E), presence of mononuclear infiltrate (Inf) and degeneration of muscle fibers (black star). (d and f) Semi-thin and ultrathin sections of a non-dengue case presenting cardiac fibers (CF) with normal nucleus (N), mitochondria (M), capillaries (Cap) and intercalated discs (ID). (e) Semi-thin section of one dengue case presenting degeneration of cardiac fibers (black star) characterized by absence of nucleus and a diffuse interstitial edema (E) and (g and h) ultrathin sections showing nuclear (white stars) and mitochondria alterations (M) in cardiomyocytes and interstitial edema. Quantitative analysis of hemorrhage (i) and edema (j) observed in dengue (cases 1–4) and non-dengue patients (cont. 1–4), and statistical analysis performed comparing the mean values of each group (dengue patients vs non-dengue patients). Asterisks indicate differences that are statistically significant between control and dengue groups, (*) (P<0.05). Semi-thin and ultrathin sections were stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083386#pone-0083386-g001" target="_blank">figure 1</a>, as well as damage quantifications. (Pt) platelets; (EC) endothelial cells.</p

Hospital and necropsy records from the four dengue fatal cases.

<p>ND: not determined.</p

Dengue virus in the heart.

<p>(a–c) Detection of DENV-3 antigens in general (a, b and c) and specifically the NS3 protein (e and f), by immunochemistry, in cardiac fibers (CF), endothelium (En) and monocytes (Mo). (d) Quantification of cells presenting dengue antigens. (g–i) Detection of virus RNA negative strand by <i>in situ</i> hybridization. Dengue cases were treated without or with the probe (g and i, respectively). (h) One non-dengue case incubated with the probe. Arrows indicate positive staining in endothelium (En) and cardiac fibers (CF).</p

Dengue virus in the spleen.

<p>Detection of DENV-3 antigens in general (a) and specifically the NS3 protein (c), by immunochemistry in macrophages (Mφ). (b) Quantification of cells presenting dengue antigens. (d–f) Detection of virus RNA negative strand by <i>in situ</i> hybridization. Dengue cases were treated without or with the probe (d and f, respectively). (e) One non-dengue case incubated with the probe. Arrows indicate positive staining in macrophages. (g) Co-localization of the virus RNA negative strand (fluorescent blue) and CD68 (fluorescent red), for identification of macrophages. Cells presenting both staining showed yellow fluorescence.</p

Dengue virus in the liver.

<p>(a–b) Detection of DENV-3 antigens in general, by immunochemistry, in hepatocytes (H), Kupffer cells (KC) and endothelium (En). (c) Quantification of hepatocytes and Kupffer cells presenting dengue antigens. (d) Detection of dengue NS3 protein by immunochemistry in hepatocytes (H), Kupffer cells (KC) and endothelium (En). (e–g) Detection of DENV-3 RNA negative strand by <i>in situ</i> hybridization. Dengue cases were treated without or with the probe (e and g, respectively). (f) One non-dengue case incubated with the probe. Arrows indicate positive staining in hepatocytes (H).</p

Detection of miR-101 in the lung of mice subjected to cigarette smoke.

<p>Mice were subjected to filtered air (FA) or cigarette smoke (CS) for 4 weeks. (A) Paraffin-embedded, formalin-fixed lung tissues were incubated with an LNA probe anti-miR-101 (purple staining), or scrambled probe as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050837#pone.0050837-Crawford1" target="_blank">[12]</a>. (B) CFTR protein (brown staining) was detected by immunohistochemistry as described in methods section. Arrows show the bronchial epithelium. The images are representative of 3–4 mice for each condition.</p

Histopathological and ultrastructural analysis of the lung.

<p>(a) Lung of a non-dengue case stained with HE and presenting normal aspect of alveoli (A) and alveolar septa (AS). (b–g) Lung sections of dengue cases, stained with HE, showing pulmonary alterations, including septal thickening (St), edema (E), hemorrhage (He), presence of mononuclear infiltrate (Inf), hyaline membrane formation (HM) and hypertrophy of alveolar macrophages (AM) and type II pneumocytes (PcyII). (h) Semi-thin section of a non-dengue case showing alveoli (A), alveolar septa (AS), endothelial cells (EC) and type I (PcyI) and II pneumocytes (PcyII) with normal aspects. (i) Semi-thin section of one dengue case presenting numerous platelets (Pt) and megakaryocytes (MK) inside alveolar septa. (j) Ultrathin section of one non-dengue case exhibiting regular alveoli, alveolar septum, type I and II pneumocytes and endothelial cell. (j-l) Ultrathin sections of dengue cases exhibited type II pneumocytes located in alveolar space in contact with numerous platelets, the appearance of hyaline membrane and the presence of virus particles (VP) in endothelium. Quantitative analysis of hemorrhage (m) and edema (n) observed in dengue (cases 1–4) and non-dengue patients (cont. 1–4), and statistical analysis performed comparing the mean values of each group (dengue patients vs non-dengue patients). Asterisks indicate differences that are statistically significant between control and dengue groups, (*) (P<0.05). Semi-thin and ultrathin sections were stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083386#pone-0083386-g001" target="_blank">figure 1</a>, as well as damage quantifications.</p

Detection of miR-101 in the lung of control (GOLD 0) and GOLD 4 COPD patients.

<p>Paraffin-embedded, formalin-fixed lung tissues from control (GOLD 0) (A&B) or patients with severe COPD (GOLD 4) (C&D) were incubated with an LNA probe anti-miR-101 (purple staining). The bronchial epithelium is shown by arrows. Images are representative of four patients per group.</p

MiR-101 targets 3′UTR of CFTR.

<p>Cells were transfected with 50 ng of psiCHECK containing wild-type (WT) or mutated (Mut) CFTR 3′UTR and 30 nM of pre-miR-101. Twenty four hours following transfection, cells were assayed for both firefly and renilla luciferase using the dual luciferase glow assay. All transfection experiments were conducted in triplicate. Data are expressed as mean±SE of at least three independent experiments. *p<0.05, n.s.: not significant.</p