Primary Acinar Cultures Primary acinar cells were prepared as previously described

lture Stimulated by Wood Smoke Condensate and Immunofluorescence Wood smoke condensate was obtained as previously described [18]. Primary rat tracheal epithelial cells (ATCC; Manassas, VA, US) were cultured in a humidified atmosphere of 5% CO2 at 37uC. Prior to the experiments, the cells were serum starved for 24 h and then stimulated with 10 mg/ml of wood smoke condensate diluted in growth medium (containing 0.3% FBS) for 7 days. Then, the cells were harvested for immunofluorescence,cell lysis and protein extraction. The cells were fixed in 4% (w/v) paraformaldehyde for 15 minutes and were then incubated with tissues were lysed in RIPA buffer (Pierce Biotechnology; Rockford, USA), the proteins were quantified using a Pierce BCA protein assay kit (Thermo Scientific; Utah, USA). Equal amounts of total protein were resolved on 8% SDS-PAGE electrophoresis. Following electrophoretic transfer, the membranes were treated at RT for 1 hour with 5% skim milk. Then, the membranes were incubated overnight at 4uC with primary antibodies against Figure 4. Increased collagen deposition in the small airways was induced by wood smoke. (a�f) Masson’s Trichrome staining and IHC staining for Type I collagen showed increased collagen deposition in the small airway wall after 7 months of CF-101 site 19653056″ title=View Abstract(s)”>PubMed ID: smoke exposure. (g, h) Graphical data showed that the area of total collagen and type I collagen deposition in the small airway wall exposed to WS or CS was significantly increased compared to controls at 7 months, but there was no significant difference at 4 months. Data are shown as the mean 6 SEM or as box and whisker plots with the median, minimum and maximum values. n = 8 animals/group. Scale bar = 50 mm. doi:10.1371/journal.pone.0096708.g004 PLOS ONE |

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