, work has received funding from the European Union, 7th Framework Programme, HEALTH-F2-2007-202224 European IPF Network, the Fonds Européen de Développement Économique et Régional, Agence Nationale de la Recherche
, Recherche en Santé Respiratoire", the Société de Pneumologie de Langue Française and the Fondation du Souffle. O. B. and P.B. are lauréat 2014 de la Fondation du Souffle et du Fonds de dotation Recherche en Santé Respiratoire. O.B., PS.B. and G.W. are supported by Fonds de Dotation « Recherche en Santé Respiratoire » et la Société de Pneumologie de Langue Française. O.B. is supported by La Ligue National Contre le Cancer. The team of C.G. has been awarded the « label d'excellence » by La Ligue National Contre le Cancer and L'Association pour la, ), and the Fonds de Dotation
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, Online supplemental methods Human tissue samples for histology Lung tissue samples were obtained by open lung biopsy (INSERM U700, Paris). IPF were diagnosed according to the
, Paraformaldehyde-fixed, paraffin-embedded sections of human lung tissue were pretreated in citrate buffer pH6 for 40 minutes for antigen retrieval. Sections were then incubated with 1B6.1-3G4 anti-?B-crystallin monoclonal antibody (Enzo Life Sciences AG, ), and with anti-calretinin antibody
, Positive cells were revealed using the Vectastain ABC-alkaline phosphatase kit system (Vector Laboratories
, The TGF-?1 cDNA has cysteine to serine mutations at position 223 and 225, rendering the expressed TGF-?1 biologically active. The control vector (AdDL) with no insert in the E1 region was produced in the same way (26) Animal procedures SV129 Wild-Type (WT) mice (Charles River, Saint Germainsur-l'Arbresle, France) and SV129 Knock-Out (KO) mice for the ?B-crystallin gene were housed in pathogen-free conditions. The adjacent gene HSPB2, which islocated in the 5' flanking region of the ?Bcrystallin gene in a head-to-head manner with an intergenic sequence of less than 1 kb,was also knocked-out in those mice (28). Mouse food and water were provided ad libitum. The animals were treated according to the guidelines of the Ministère de la Recherche et de la Technologie, The construction of adenoviral vectors has been described previously (26, 27). Briefly, TGF-?1 cDNA was cloned into a shuttle vector downstream a human CMV promoter and cotransfected with a shuttle plasmid in 293 cells
To induce pleural/subpleural fibrosis, adenovectors and bleomycin/carbon particles administrations were performed by intrapleural injection of 100 µl of sterile NaCl 0 ,
Adenovectors were administered, without any surgery, by intrapleural injection on the right side (6th intercostal space) with a 20-gauge needle, with animals in a left lateral decubitus position. Pleural lavage fluid (PLF) was collected as previously described (29) and stored at-20°C after cell count. ?-galactosidase staining ?-galactosidase staining was performed as previously described (7). Cytochemical staining for ?-galactosidase was performed on samples obtainedfrom animals after intrapleural injection of AdLacZ/AdTGF-?1 or AdLacZ/AdDL. After 1 h in fixative (2% ferrous cyanide,potassium ferric cyanide, magnesium chloride ,
Samples were stored in 70% ethanol and then paraffin-processed and-embedded ,
, Five-micrometer sections were counterstained with nuclearfast red. Collagen quantification (sircol and histomorphometry)
, Histomorphometric assay-The amount of collagen in paraffin-embedded tissue sections was quantified by staining with Picrosirius Red. The percentage of emission (reflecting the percentage of collagen) of the pleural, subpleural and parenchymal areas were quantified (morphometry software from Histolab/Microvision Instruments, Evry, France) as previously described(8). Briefly, collagen intensity in the pleura was measured within a rectangle (constant length of100 mm
, Cells were then transfected with ?B-crystallin siRNA or a Scramble SiRNA and treated rTGF-?1 for 48h or left untreated. Pictures were taken immediately after the scratch and 48h after rTGF-?1 treatment (Zeiss,Oberkochen, Germany), Undergraduate vocational diploma. Institute of Technology, 2007.
, MRes-UMR INSERM U866-« Lipids, 2012.
France Role of HSP27 in pulmonary fibrosis and in epithelial-to-mesenchymal transition-Language : French: mother tongue, English: fluent, German: basic knowledge-Certificate for animal experimentation (FELASA C level equivalent), Softwares : office, flow cytometry software (Cellquest, Diva, FlowJo) Publications, 2011. ,
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