Fig. 6

TGF-β1 caused a decrease in catalase mRNA and protein levels. A, B The immunoreactivity of catalase was lower, and that of GPX1/2 higher in IPF (right) compared to control (left) lung tissues. Immunofluorescence staining was performed using antibodies to detect catalase (A, red) and GPX1/2 (B, red) and DAPI to counterstain nuclei. C The protein level of catalase is lower in IPF than in control fibroblasts. Cell lysates of fibroblasts from 5 control and 5 IPF patients were used for Western blot analysis of catalase (CAT) with β-actin (β-ACTIN) as reference protein. D TGF-β1 decreased catalase protein levels in control and IPF fibroblasts. Cells were serum-starved for 3 h, and treated with various concentrations of TGF-β1 or vehicle for 48 h. Cell lysates were used for Western blot analysis of catalase with GAPDH as reference protein. E–G Activation of PPAR-β/δ in combination with PPAR-γ restored TGF-β1-induced decrease in catalase protein levels and activity. E TGF-β1 decreased catalase activity in control and IPF fibroblasts. Cells were serum-starved for 3 h, and treated with vehicle (Control) or various concentrations of TGF-β1 for 12 h. Cell lysates were used for measuring catalase activity. F, G Activation of PPAR-β/δ in combination with PPAR-γ inhibited TGF-β1-induced decrease in catalase protein levels in control and IPF fibroblasts. Cells were serum-starved for 3 h, stimulated with vehicle (F, G) or TGF-β1 (5 ng/ml, F, G) or for 24 h, followed by the addition of the PPAR-β/δ agonist GW0742 (10 μM, β) and the PPAR-γ agonist rosiglitazone (10 μM, γ) for another 24 h (F). In (G), the PPAR agonists were added together with TGF-β1 for 48 h. Cell lysates were used to detect catalase (CAT) by Western blot analysis using α-tubulin (α-TUB) as reference protein