Fig. 7

Catalase contributes to collagen reduction in pulmonary fibrosis. A, B Stable knockdown of catalase decreased catalase protein and activity. Cell lines transfected with catalase shRNA (CAT sh1, CAT sh2) were serum-starved for 3 h. Cell lysates were used for measuring catalase (CAT), COL1 and α-SMA protein levels by Western blot analysis using GAPDH as reference protein (A) and catalase activity by catalase activity assay kit (B). C, D Stable knockdown of catalase increased the cellular H₂O₂ production and extracellular collagen levels. Culture media from catalase-deficient (CAT sh1, CAT sh2) and mock-transfected (CAT sc) control fibroblasts were used to detect the release of H₂O₂ using the hydrogen peroxide assay (C) and of extracellular collagen by Sircol assay (D). E Overexpression of catalase decreased the protein level of COL1 in control and IPF fibroblasts under basal condition (no treatment) and after TGF-β1 treatment. Control and IPF fibroblasts were transfected with pGL 4.14-Catalase (CAT overexpr.) or a mock vector for 48 h, followed by the addition of vehicle or TGF-β1 (5 ng/ml) for another 48 h. Cell lysates were analyzed for catalase (CAT), α-SMA, and COL1 protein levels by Western blot analysis using GAPDH as reference protein. F The catalase activity inhibitor AT does not increase COL1 in control and IPF fibroblasts. Cells were serum-starved for 3 h, treated with vehicle or TGF-β1 (5 ng/ml) or for 24 h, followed by the addition of the PPAR-β/δ agonist GW0742 (10 μM, β), the PPAR-γ agonist rosiglitazone (10 μM, γ) and AT (25 µM) as well as various combinations thereof for another 24 h. Cell lysates were used to analyze catalase (CAT), COL1, and α-SMA protein levels by Western blot analysis using GAPDH as reference protein