Fig. 4

TGFβ regulates E-Cadherin, Vimentin and Collagen I on protein level in hAECII. Immunocytochemistry (a) was used to assess protein expression of E-Cadherin, Vimentin and Collagen I in paraffin-embedded hAECII from cell culture experiments (N = 2). Representative image shown with a scale bar = 100 μm. Red colour indicates positive signals. b-e Double immunofluorescence staining was used to show an up-regulated collagen metabolism in AECII lineage by TGFβ-stimulation. The thyroid transcription factor I (TTF-1) of the surfactant molecules was targeted as a proof of AECII lineage and the collagen chaperon HSP47 was used to display an elevated collagen synthesis. Nuclei are stained by DAPI (blue). TTF1 was visualized with a TRITC-conjugated secondary antibody (red) and HSP47 with an Alexa488-conjugated (green) secondary antibody. Exemplary image of 3 biological replicates with scale bar =50 μm show the expression of HSP47 in medium control b, with 5 ng/ml TGFB1 C, with 10 μm SB431542 (d) or both, TGFB1 and SB431542 (e). For means of cross-validation of results from ICC and IF, In-Cell Western analysis was conducted (f)s to investigate regulation of Collagen I, HSP47, N-Cadherin and ZO1 on protein level in hAECII . Cells were seeded in 96 well plates and stimulated for 48 h. Protein expression of Collagen I, HSP47, ZO1 and N-Cadherin/CDH2 was detected by means of ICW assay with primary antibodies against the targets and near-infrared conjugated secondary antibodies. To-Pro3 was used to stain all cells as loading control. Data is shown as semi-quantitative fluorescent signal normalized to total amount of cells, corrected for background signal and normalized to medium controls. N = 5 (HSP47, ZO1, N-Cadherin) or N = 9 (Collagen I). Semi-quantitative measurement of collagen produced by hAECII as stained by SiriusRed (N = 7)