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Fig. 6 | Respiratory Research

Fig. 6

From: C-type natriuretic peptide ameliorates pulmonary fibrosis by acting on lung fibroblasts in mice

Fig. 6

CNP attenuated TGF-β-induced fibroblast differentiation in human lung fibroblasts. Primary cultured human lung fibroblasts (LF) from a surgically resected specimen of a lung cancer patient (a) and its immortalized and GC-B stably expressed cell-line (LFhTERT/GC-B) (b) were treated with CNP at 10 nM to 1 μM in combination with 3-isobutyl-1-methylxanthine (0.5 mM). The cGMP concentrations in the cell lysate were measured 15 min after CNP-treatment. Relative concentrations of cGMP (mean of control without CNP = 1) are shown. Values represent means ± SEM. c Gels comprising type I collagen (0.75 mg/ml) and LFhTERT/GC-B cell suspension (5 × 105 cells/ml) were maintained in serum-free DMEM with or without 1 ng/ml TGF-β. CNP was added to the medium at a final concentration of 1 μM every 24 h. After 3 days, the gels were released from the plate and their diameter was measured. The percentage of contraction is shown. Values represent means ± SEM. *P < 0.05. Results are representative of three independent experiments. d and e The relative mRNA expression of α-SMA (d) and SM22α (e). LFhTERT/GC-B cells were serum-starved for approximately 24 h. The cells were then pretreated with CNP (1 μM) in DMEM + 1 % FCS for 30 min and treated with or without TGF-β (1 ng/ml) for 24 h (for mRNA analysis) or 48 h (for protein analysis). Expression levels were normalized to that of 36B4 mRNA, and then relative expression levels were calculated (mean of control = 1). Values represent means ± SEM. *P < 0.05. Results are representative of three independent experiments. f Immunoblot analysis of α-smooth muscle actin (SMA), SM22α, fibronectin, connective tissue growth factor (CTGF), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cells were treated as in (d). Then cell lysates were prepared and subjected to immunoblot analysis. A representative blot of three independent experiments is shown. g The LFhTERT/GC-B cells were serum-starved for approximately 24 h. The cells were then pretreated with CNP (1 μM) in DMEM + 1 % FCS and treated with or without TGF-β (1 ng/ml) for 30 min. Cell lysates were immunoblotted for phospho-Smad 2 (p-Smad2) or total Smad 2 (t-Smad2). A representative blot of three independent experiments is shown with the ratio of the p-Smad2 to t-Smad2 signal intensities

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