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Fig. 1 | Respiratory Research

Fig. 1

From: Proteases and oxidant stress control organic dust induction of inflammatory gene expression in lung epithelial cells

Fig. 1

Protease activities in dust extract and the effects of protease inhibitors and heating on IL-8 mRNA and protein levels. a Trypsin and elastase activities in dust extract were measured using BAPNA and SAPNA substrates, respectively. Dust extract (5 μl) was mixed with BAPNA (0.92 mM) or SAPNA (0.37 mM) in a final volume of 200 μl of 0.1 M Tris-HCl 8.0 or 0.1 M Tris-HCl 8.3, incubated at room temperature and absorbance at 410 nm recorded at indicated times. Data shown are average of duplicate measurements. Similar results were obtained in a second independent experiment. b and c Trypsin and elastase activities were measured in the presence of protease inhibitor cocktail (0.5 ×) or α1-antitrypsin (10 μg) (α1-AT). Data shown are means ± SD of two independent experiments. d A549 cells were treated with medium (C), dust extract (0.25 %) (DE), dust extract (0.25 %) that was heated at 95 °C for 10 min, or dust extract (0.25 %) in the presence of 2 μl protease inhibitor cocktail (PIC), 10 μg/ml α1-antitrypsin (α1-AT), or 10 μg/ml soybean trypsin inhibitor (SBTI) for 3 h and IL-8 mRNA levels determined by qRT-PCR. IL-8 mRNA levels in dust extract treated cells were arbitrarily considered as 100, and relative IL-8 mRNA levels in other treatments are shown. Data shown are means ± SE (n = 3). **P < 0.01; ***P < 0.001. e A549 cells were treated with medium (C), dust extract (DE) (0.25 %), dust extract (0.25 %) heated at 95 °C for 10 min, or dust extract (0.25 %) in the presence of 25 μg/ml α1-antitrypsin (α1-AT), 25 μg/ml soybean trypsin inhibitor (SBTI), or 10 μM E64 for 3 h. IL-8 levels in cell medium were determined by ELISA. IL-8 levels in dust extract treated cells were arbitrarily considered as 100, and relative levels in other treatments are shown. Data are means ± SE (n = 3–6). *P < 0.05, # P < 0.0001

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