Fig. 4

Nicotine Stimulates Collagen Expression in Lung in vivo. a The lungs of mice exposed to nicotine (100 μg/ml in the drinking water for 8 weeks) were isolated for RNA analysis, which showed an increase in collagen type I expression in nicotine-treated wildtype mice, but not in α7KO mice. b Protein analysis showed increased p-ERK 1/2, p-Smad3, and collagen type I protein expression when compared to control lungs. P-Erk, p-Smad3, and collagen type 1 were all completed on the same gel, with the blot stripped between probings. The total Erk, Smad3 gels were run on a separate gel and stripped between probings. c Lungs were also inflated at standard pressure, fixed in formalin, paraffin-embedded, and sectioned (5 μm) for histological analysis. Lung sections were stained using Weigert's iron hematoxylin for 10 min., rinsed, treated with Biebrich scarlet-acid fuchsin solution for 10 min., washed and transferred to aniline blue stain for 30–60 min or stained with Sirius Red/Fast Green for 30 min. Arrows indicate increased collagen type I deposition (blue stain or Red stain) in wildtype animals exposed to nicotine. d The tri-chrome slides were blindly graded on a scale of 0–3 based on the intensity of collagen staining. e Nicotine treatment resulted in no differences in tri-chrome staining in α7KO mice. Experiments were repeated at least 3 times. Significance was assessed using p values <0.05 obtained by two-tailed t-test