Figure 1.
Induction of alveolar enlargements in mice with targeted expression of mutant B-RAF (V600E) in lung alveolar type II cells.
(A) Schematic representation of the targeting vector. (B) Semi-quantitative RT-PCR shows transgenic- and endogenous- B-RAF expression in lungs of two wild type and transgenic embryos (E16.5), (cycles = PCR cycle; M = marker; h = human; m = mouse). (C) Paraffin embedded hematoxylin and eosin (H&E) stained lung sections from mice of different ages show airspace enlargements (arrows) in transgenic animals, (scale bar = 100 µm for the first, second and third panel; 1 mm for the forth panel). (D) Whole lung photographs of 4.5 months old wild type and transgenic mice show a macroscopic bleb (arrows). (E) Kaplan-Meier survival curve, (log-rank analysis P<0.05; SpC-B-RAF V600E n = 59; wild type n = 30).
Figure 2.
Immunohistochemical analysis of lung lesions for lung differentiation markers.
Paraffin embedded lung sections from wild type and transgenic mice at 2 months of age were stained for indicated markers; (green arrows point out airspace enlargements), (the red arrow marks the bronchio-alveolar junction), (red-parenthesis indicates a AQP5 negative sector), (red arrowheads point to Pro-SPC expressing cells), (black arrows highlight Ki67 positive cells), (Br = bronchiole), (hematoxylin was used as a counterstain), (right panel pictures are high magnification of the red insets); scale bar = 100 µm.
Figure 3.
Analysis of cell death and cell proliferation in transgenic mice.
(A) Active caspase 3 staining of control and transgenic animals, (arrows indicate positive cells). (B) Ki67 staining (brown) of control and transgenic animals. (E = embryonic, P = post-natal), (hematoxylin was used as a counterstain); scale bar = 50 µm.
Figure 4.
Collagen accumulation and Goblet cells hyperplasia in aged SpC-B-RAF V600E transgenic mice.
(A) Masson's Trichrome staining shows collagen (blue), red-parenthesis indicates Masson's Trichrome negative parts, (the right panel pictures are high magnification of the red insets). (B) Representative pictures of Alcian blue stained paraffin embedded lung sections from wild type and transgenic mice at 3 and 12 months of age, respectively, black arrowheads denote Alcian Blue-positive (blue) regions indicative of increased mucus production; scale bar = 100 µm.
Figure 5.
Analysis of Epithelial-Mesenchymal-Transition (EMT) in SpC-B-RAF V600E transgenic mice.
(A) Total lung protein lysates from two wild type (wt) and transgenic (tg) littermates (17 and 21 months old) were gel separated and immunoblotted with the indicated antibodies. (B) Immunostaining of lung sections from wild type and SpC-B-RAF V600E transgenic mice for Vimentin (brown), (E = airspace enlargement), hematoxylin was used as a counterstain, scale bar = 100 µm. (C) Analysis of E-cadherin and Vimentin mRNA levels in alveolar type II cells isolated from wild type and SpC-B-RAF V600E transgenic mice (5 months old) by Real time PCR, data represent mean+SEM, (t-test * = P<0,05, ** = P<0,01, n = 3–4). (D) Protein lysates of isolated type II cells from two wild type (wt) and transgenic (tg) littermates (20 months old) were gel separated and immunoblotted with the indicated antibodies.
Figure 6.
Progressive accumulation of macrophages in the lung of SpC-B-RAF V600E transgenic mice.
Paraffin embedded lung sections from mice with the indicated genotypes and ages stained for F4/80 (brown), E = airspace enlargement, arrowheads show diffused alveolar macrophages, hematoxylin was used as a counterstain, scale bar = 100 µm.
Figure 7.
Infiltration of immune cells in B-RAF V600E-induced lung lesions.
(A) Toluidine blue staining identifies mast cells (purple) in the epithelial lining of a lesion. (B) Staining of paraffin embedded lung sections from wild type and transgenic mice for CD45 (brown), hematoxylin was used as counterstain, the right panel pictures are high magnification of the red insets; scale bar = 100 µm.
Figure 8.
Activation of STAT3 signaling in SpC-B-RAF V600E transgenic mice.
(A) Total lung protein lysates from two wild type (wt) and transgenic (tg) littermates (17 and 21 months old) were gel separated and immunoblotted with the indicated antibodies. (B) Phospho-STAT3 immunostaining of lung sections shows accumulation of STAT3 positive cells in transgenic mice (arrows indicate a macroscopic bleb, arrowheads point to Phospho-STAT3 positive cells), the right panel pictures are high magnification of the red insets, hematoxylin was used as a counterstain, scale bar = 1 mm in the right and 100 µm in the left panel pictures.
Figure 9.
Lung tumor formation in SpC-B-RAF V600E transgenic mice.
(A) Tumor incidence as a function of age, log-rank analysis for wild type (n = 59) and SpC-B-RAF V600E (n = 102) transgenic mice, P<0.05. (B) H&E staining of lung section from a one year old SpC-B-RAF V600E transgenic mouse shows coexistence of the lung lesions (arrowheads) with a lung tumor (arrow); scale bar = 500 µm. (C) Tumor bearing lung sections were stained with the indicated markers, hematoxylin was used as counterstain; scale bar = 100 µm.