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

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

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