Fig. 9

Representative immunofluorescence images for the evaluation of oxidative stress, remodelling pathway, and fibrosis markers specific to vascular dysfunction, lung and cardiac fibrosis after 12 weeks of MCT alone or in combination with siRNA AP-1 for 10 weeks. The thin cryo-sections from three types of tissues: pulmonary artery, lung and right ventricle harvested from all experimental groups (C, MCT, MCT-siRNA AP-1) were immuno-labelled for: A Cytosolic ROS (Reactive Oxygen Species) production stained with dihydroethidium (DHE) dye shown in bright red nuclear fluorescence; Bar graph with quantification of the stained areas expressed in: (a) pulmonary artery, (b) lung, (c) right ventricle, (d) liver. B proteins involved in vascular remodelling/fibrosis: collagen type I (COL1A1-red), Fibronectin-red, Matrix metalloproteinase-9 (MMP-9-green); Bar graph with quantification of the stained areas expressed in pulmonary artery. C proteins involved in vascular fibrosis and the epithelial-mesenchymal transition (EMT): Connective tissue growth factor (CTGF-red), Calponin-red, Vimentin-red, alpha smooth muscle actin (α-SMA-green); Bar graph with quantification of the stained areas expressed in pulmonary artery. D specific endothelial markers for EMT assessment: PECAM-1 (CD31-red), Vascular endothelial cadherin (VE-cadherin-green); Bar graph with quantification of the stained areas in pulmonary artery. E proteins involved in pulmonary fibrosis and EMT: COL1A1-red, CTGF-red, Fibronectin-red, α-SMA-green; Bar graph with quantification of the stained areas expressed in lung tissue. F, G proteins involved in cardiac hypertrophy/fibrosis and FMT: COL1A1-red, CTGF-red, Fibronectin-red, α-SMA-red, F-actin (Phalloidin-green) OB-cadherin-red; Bar graph with quantification of the stained areas expressed in right ventricle tissue. Nuclei were stained with DAPI dye shown in blue fluorescence. Data were presented as mean ± SD. Each experiment point was performed in triplicate, from two different set of experiments. Five different microscopic fields for each experimental point were analysed. 20 × magnification, images quantified using the ImageJ program. The statistical significance, noticeably different, was represented as ***P < 0.005, **P < 0.01, *P < 0.05 values versus control group and ^###P < 0.005, ^##P < 0.01, ^#P < 0.05 values versus MCT group. One-way ANOVA, Bonferroni post-test was applied