Figure 2: Active TGF-β1 and collagen expression are increased in PTENMyKO mice in early fibrosis.
7 days post BLM treatment the lungs of myeloid PTEN deficient and wild-type mice lungs were removed and homogenized either for ELISA and RNA isolation.(a)In vivo active (cleaved) TGF-β1 was measured by ELISA and found to be increased in myeloid PTEN deficient mice. (b) The same ELISA was performed after activating the samples with HCl in vitro for measuring latent TGF-β1 together with active TGF-β1. For statistical analysis the Student’s T-test was performed, n = 12–15, ***p < 0.001; (c)SMAD2/3 phosphorylation 7 days post BLM treatment. Broncho-alveolar lavage cells were analysed by Western blot. (d) Densitometric analysis of the protein content of the Western blot, the data are presented as fold of β-Actin and normalized to control mice, n = 2. (e–l) Lungs of BLM treated mice after 7d and 21d were homogenized for RNA isolation and qPCR was performed. The following genes were determined: collagen, type I, alpha2 (col1a2), collagen, type I, alpha 1 (col1a1), collagen, type III, alpha1 (col3a1), lysyl oxidase (lox), tgfb1, alpha smooth muscle actin (acta2), tissue inhibitor of metalloproteinase 3 (timp3), integrin alpha V (itgav)and hypoxanthine guanine phosphoribosyl transferase (hprt). Expression levels were normalized to HPRT as housekeeping gene. Data are shown as fold values of BLM treated wild-type (light bars) and PTENMyKO (grey bars) mice relative to treatment-naive mice. For statistical analysis the Mann Whitney test was performed, n ≥ 4 (7d), n ≥ 2 (21d), *p < 0.05
