Immunostaining of histological sections of Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i> and CMV-<i>Cre</i>^T/+; <i>Kras</i>^+/LSLG12Vgeo adenomas (<i>Kras</i>^+/LSLG12Vgeo 6 months) and Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i>; <i>Lkb1</i>^flox/+ and CMV-<i>Cre</i>^T/+; <i>Kras</i>^+/LSLG12Vgeo adenocarcinomas (<i>Kras</i>^+/LSLG12Vgeo 11 months) with CC10, SP-C, E-Cadherin, Ki67, p53 and LKB1 antibodies.

<p>(*) indicates airways. Bars 500 µm and 80 µm for magnifications.</p

Neonatal activation of BRAF^V600E through the expression of Tyr::<i>Cre</i>^ERT2 upon 4OHTx treatment drives aberrant proliferation of lung cells.

<p>(<b>A</b>) Schematic representation of mouse treatment and expressed proteins. (<b>B</b>) Representative images of Cre-recombinase staining in 4-days-old wild type (WT, n = 3) and Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i> (n = 3) mice lungs treated with 4OHTx. Bars 80 µm. (<b>C</b>) Anti-p-ERK1/2 staining of untreated (−4OHTx) and treated (+4OHTx) 4-days-old Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i> mice lungs. (<b>D</b>) Schematic representation of the genetic strategy to identify tyrosinase-promoter driven Cre-recombinase lung expressing cells. Representative images of EYFP, SP-C and CC10 expressing cells in Tyr::<i>Cre</i>^ERT2;ROSA-lsl-<i>EYFP</i> mice 3 days after 4OHTx treatment (n = 3 mice) are shown. Bars 80 µm. (<b>E</b>) Ki67 staining of histologically normal lungs in 8-days-old mice showed increased proliferation index in 4OHTx treated Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i> mice compared to untreated. Bars 500 µm. Quantification of samples is shown below. 20X fields (n = 8 and n = 11 from 3 different untreated and 4OHTx treated mice respectively) were quantified. <i>p</i>-value was calculated performing Mann-Whitney’s test.</p

Percentage of mice developing lung adenomas (L. adenomas) or lung adenocarcinomas (L. carcinomas) according to their genotype and treatment (4OHTx).

<p>Percentage of mice developing lung adenomas (L. adenomas) or lung adenocarcinomas (L. carcinomas) according to their genotype and treatment (4OHTx).</p

Percentage of mice developing adenomas and adenocarcinomas.

<p>Hematoxylin and eosin staining of histological sections showing lung hyperplasia in Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i>; <i>Lkb1</i>^flox/+ (<b>A</b>, <b>B</b>) and CMV-<i>Cre</i>^T/+; <i>Kras</i>^+/LSLG12Vgeo mice (<b>J</b>, <b>K</b>). Higher magnification is showed in (<b>B</b>, <b>K</b>). Papillary adenomas developed in Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i>; <i>Lkb1</i>^flox/+ (<b>C</b>, <b>D</b>) and mixed papillary and solid adenomas developed in CMV-<i>Cre</i>^T/+; <i>K-ras</i>^+/LSLG12Vgeo (<b>L</b>, <b>M</b>). Note <b>C</b> and <b>L</b> tumors in higher magnification (<b>D</b>, <b>M</b>). Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i>; <i>Lkb1</i>^flox/+ adenocarcinoma (<b>E</b>) showing papillary (<b>F</b>) and solid (<b>G</b>) regions. Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i>; <i>Lkb1</i>^flox/+ adenocarcinoma showing intra bronchiolar tumor growth (*) (<b>H</b>). Higher magnification showing different cells populations in <b>H.</b> Atypical cells with nuclear hyperchromasia, and contour irregularities (*), cells showed enlarged nuclei displaying prominent nucleoli (**) and cells with hyperchromatic fusiform nuclei (arrows). CMV-<i>Cre</i>^T/+; <i>Kras</i>^+/LSLG12Vgeo adenomas and adenocarcinomas (<b>N</b>). Detail of solid (<b>O</b>) and mucinous (<b>P</b>) tumors. Dashed-lined squares indicate magnified areas. Bars 800 µm (<b>A</b>, <b>C</b>, <b>D</b>, <b>J</b>, <b>L</b> and <b>N</b>), 500 µm (<b>E</b>), 200 µm (<b>K</b>, <b>M</b>, <b>O</b> and <b>P</b>) and 100 µm (<b>B</b>, <b>D</b>, <b>F</b>, <b>G</b> and <b>I</b>).</p

Neonatal activation of BRAF^V600E in Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+ mice</i> promotes lung adenomas development.

<p><b>(A)</b><b> </b> Kaplan-Meier analysis of lung tumor-free survival in untreated and 4OHTx treated Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i> and Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/CA</i> mice. <i>p</i>-value was calculated by Logrank Test. On the right, percentage of mice developing lung adenomas is shown (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066933#pone-0066933-t001" target="_blank">Table 1</a>). (<b>B</b>, <b>C</b>) Hematoxylin and eosin staining of histological sections of Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i> bronchiolo-alveolar adenoma or (<b>D</b>, <b>E</b>) normal lung from wild-type mice. Note papillary adenomas and normal lung in higher magnification (<b>C</b>, <b>E</b>). Adenomas stain negative for CC10 (<b>F, G</b>) and positive for SP-C (<b>H</b>, <b>I</b>). Bars 500 µm (<b>B</b>, <b>D</b> and <b>F</b>), 300 µm (<b>C</b> and <b>D</b>) and 100 µm for (<b>G</b> and <b>I</b>).</p

Percentage of mice developing lung adenomas (L. adenomas) or lung adenocarcinomas (L. carcinomas) according to their genotype and treatment (4OHTx).

<p>Percentage of mice developing lung adenomas (L. adenomas) or lung adenocarcinomas (L. carcinomas) according to their genotype and treatment (4OHTx).</p

Effect of MTA on liver myofibroblast intracellular signalling pathways.

<p>Liver myofibroblasts were pre-treated with MTA for 30 min in the absence of serum and then stimulated with 10% FCS for 30 min or PDGF (20 ng/ml) for 60 min. Phosphorylation levels of Erk1/2 and c-Jun upon FCS stimulation (A), and S6 ribosomal protein after PDGF treatment (B) were analyzed by western blotting. Representative blots are shown.</p

Potential mechanisms of action of MTA in <i>Mdr2^−/−</i> mice.

<p>Our <i>in vivo</i> and <i>in vitro</i> observations indicate that MTA displays different actions that may underlie its beneficial effects on the course of liver injury and fibrosis in <i>Mdr2^−/−</i> mice. MTA may exert a direct cytoprotective effect on hepatocytes by reducing JNK activity, preventing hepatocellular death and further inflammation. MTA also inhibits the production of cytokines by inflammatory cells, likely through interference with NFκB activity as described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015690#pone.0015690-Hevia1" target="_blank">[16]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015690#pone.0015690-IglesiasAra1" target="_blank">[18]</a>. In addition, enhanced expression of adenosine A2B receptors may contribute to the anti-inflammatory pharmacological profile of MTA. This compound can also exert direct effects on extracellular matrix (ECM) producing cells (myofibroblasts), reducing their activation, proliferation and the production of pro-fibrogenic factors. Inhibition of JunD expression may be an important event in the antifibrogenic action of MTA.</p

Effects of MTA on liver myofibroblast activation and cytokine expression.

<p>Primary myofibroblasts were serum starved (12 h) and then treated (12 h) with 10% FCS and MTA as indicated. mRNA levels of α1(I)procollagen (A), αSMA (B), TGFβ1 (C), TGFβ2 (D), Mcp-1 (E) and IL6 (F) were measured at the end of treatments. *<i>P</i><0.05 <i>vs</i> FCS, #<i>P</i><0.05 <i>vs</i> FCS+MTA 200 µM.</p

MTA modulates intracellular signalling pathways activated in <i>Mdr2^−/−</i> mouse liver.

<p>Phosphorylation levels of Smad1/5/8 (A), Smad2 (B), JNK and c-Jun (C) and Erk1/2 (D) were analyzed by western blotting in liver extracts from <i>Mdr2^+/+</i>, control <i>Mdr2^−/−</i>, and MTA-treated <i>Mdr2^−/−</i> mice. *<i>P</i><0.05 <i>vs Mdr2^+/+</i> mice, #<i>P</i><0.05 <i>vs</i> untreated <i>Mdr2^−/−</i> mice.</p