TMPRSS4 reduces alveolar epithelial growth rate.

<p>Panel A: Human alveolar epithelial cells (A549) were cultured in 48-well culture plates in medium without FBS and stimulated with TMPRSS4 (0.1, 1, 10 and 100 ng/mL). Panel B: The analysis was repeated with 100 ng/ml in two independent experiments. Cell numbers were estimated by WST-1 assay at 4 days of culture. Each point represents means ± SD of one representative experiment performed in triplicate. *P < 0.05.</p

TMPRSS4 is expressed by epithelial and mast cells in bleomycin-induced pulmonary fibrosis.

<p>Representative photomicrographs of immunohistochemical staining performed with antibody against TMPRSS4 in lung tissue sections from WT mice injured with bleomycin. The immunoreactive enzyme was observed in epithelial (panel A, black arrow) and interstitial cells (panels B and C black arrows). Panel D: no positive staining was detected in normal lungs. Panel E: shows the negative control where the specific antibody was omitted. All sections were counterstained with hematoxylin. Panels F-I: Representative images of immunofluorescence staining performed with specific antibodies against TMPRSS4 and tryptase; Tissues were stained for TMPRSS4 (Dylight-549, red) and tryptase (AF-488, green). The colocalization of TMPRSS4 and tryptase was determined by fluorescence microscopy and images were merged to resolve the co-localization of these proteins.</p

TMPRSS4 increases E-cadherin expression.

<p>Human alveolar epithelial cells (A549) were stimulated with TMPRSS4 (100 ng/mL) or TGF-β1 (5 ng/mL) and the expression of E-cadherin and α-SMA was determined by quantitative RT-PCR and Western blot. Panel A and C: At 4 days, TMPRSS4 significantly increased the level of E-cadherin mRNA and reduced α-SMA mRNA compared with control sample (*p <0.01). Data were normalized to the level of 18S rRNA. Panel B and D: Total protein from stimulated cells was extracted and western blot analysis performed with specific antibodies for E-cadherin and α-SMA. TGF-β1 was used as a positive control for EMT. Panel E: Rat (RLE-6TN) alveolar epithelial cells were stimulated with TMPRSS4 (100 ng/mL) or TGF-β1 (5ng/mL) for 4 days. Westerns are representative of three independent experiments. Panel F: Western blot analysis of control A549 epithelial cells and A549 stimulated with TGFβ1, TMPRSS4 or TGFβ1 plus TMPRSS4.</p

TMPRSS4 is expressed by alveolar and bronchial epithelial cells.

<p>Panel A: Gene levels of TMPRSS4 were quantified by real-time RT-PCR in A549 and HBE4-E6/E7 epithelial cells and in normal (n = 3) and IPF lung fibroblasts (n = 3). Expression was observed in both epithelial cell lines while no expression was found in fibroblasts. Panel B: Protein expression of TMPRSS4 (48 KDa) was confirmed by western blot in both epithelial cell lines. Panel C: Representative Western blot of TMPRSS4 in normal and IPF lung fibroblasts. A549 epithelial cells were used as positive control. Panel D: Western blot of A549 epithelial cells stimulated with TGFβ1 for 96 hours.</p

Immunolocalization of TMPRSS4 in IPF and control lungs.

<p>Representative photomicrographs of immunohistochemical staining performed with antibody against TMPRSS4 in lung tissue sections. Strong staining was observed in epithelial (panels A and B) and interstitial cells (panel C) in IPF lungs (n = 5), whereas no positive labeling was detected in normal lungs (n = 3) (panel D). Panel E shows the negative control where the specific antibody was omitted. All sections were counterstained with hematoxylin. Arrows indicate positive cells.</p

TMPRSS4 deficiency reduces bleomycin-induced collagen deposition in lungs.

<p>Wild type (WT), TMPRSS4 deficient (KO) and haplodeficient (HT) mice were instilled with Bleomycin (7 U/Kg) and studied at 28 days. Collagen content was quantified by hydroxyproline assay. Data are expressed as mean ± SD of 7–12 animals per group. *<i>P</i>< 0.01 compared with WT instilled animals.</p

TMPRSS4 is upregulated in idiopathic pulmonary fibrosis (IPF) lungs.

<p>Gene expression of TMPRSS4 was quantified by real-time PCR in total RNA obtained from IPF (n = 7), hypersensitivity pneumonitis (HP) (n = 6) and normal lungs (n = 4). Strong upregulation was observed in IPF tissues compared with HP and control lungs. Data are expressed as means ± SD of copy number normalized to 18S rRNA; * p<0.05, IPF versus control and HP.</p

TMPRSS4 deficiency attenuates bleomycin-induced lung damage in mice.

<p>Wild type (WT), TMPRSS4 deficient (KO) and haplodeficient (HT) mice were instilled intratracheally with bleomycin (7 U/Kg) or saline solution and studied at 28 days. Panel A: control mice instilled with saline solution. Panel B: mice with bleomycin-induced pulmonary fibrosis. Histopathologic analysis was performed using hematoxylin and Masson trichrome staining.</p

Tumorigenesis and DNA transfer in recipient murine NIH3T3 cells after passive transfection. A.

<p>Tumor growth in nude mice from “passively” transformed cells. Faster and higher tumor growth was observed in SB1 pool and CCPS pool (NIH3T3 exposed to supernatant of SW480 cells and to the serum of a patient with colon cancer, respectively). SW480 cells were used as positive control, whereas NIH3T3 and NIH3T3 exposed to normal serum showed essentially no growth<b>. B.</b> Representative pictures of tumors in mice from each group. <b>C.</b> Southern blot hybridization of SB1 and CCPS pools of cells against genomic DNA of SW480 cells. Lane SW480 cells are the positive control and NIH3T3, the negative one. A clear hybridization signal is only observed in SB1 and CCPS lanes. <b>D.</b> FISH analysis of repetitive human sequences. Positive control is human lymphocytes and murine cells negative control. SB1 cells shows strong signal. <b>E.</b> Tumor growth is similar in NIH3T3 actively transfected with genomic DNA from SW480 cells (Neo-Geno) and actively transfected DNA extracted from supernatant of SW480 cells as compared with no growth in NIH3T3 (-Crt) and transfected with the empty-vector only. Positive control, SW480 cells.</p

Human DNA transfer in rat colon tumors by PCR-sequencing.

<p>Representative pictures of PCR detection of a repetitive sequence of rat (<i>LINE 1</i>) in a rat tumor (DMH and DMH+SW480). (<b>A</b>) <i>Alu Yd6</i> human sequences were only amplified from the colon tumors of DMH+SW480-treated rats. Rat tail and human cells were used as positive and negative controls. Human <i>K-ras</i> and <i>RAB30</i> genes were only detected in the tumors of rats receiving DMH and SW480 cells. (<b>B</b>) Sequence analysis of the PCR product of <i>RAB30</i> in a colon tumor treated with DMH+SW480 cells. Arrows indicate the position where the nucleotide sequence is different between species and clearly shows the existence of both sequences. Human SW480 cells (control).</p