Fig. 1.

Nicotine Stimulates Collagen Type I mRNA and Protein Expression. a Primary lung fibroblasts (1 × 10⁶ cells/6 well) were treated with nicotine (1–75 μg/ml) for 24 h. Real-time PCR reactions were performed using mouse collagen type I or 18S primers. Note that nicotine induced a dose-dependent increase in collagen type I mRNA expression. mRNA levels were normalized to 18S and collagen type I levels compared to untreated controls. b Nicotine-treated fibroblasts were subjected to Western blot analysis using anti-collagen type I antibody or GAPDH, followed by secondary goat anti-rabbit IgG (IRDye®). Protein bands were separated by native (collagen type 1) or SDS-PAGE (GAPDH) gel electrophoresis (8%) and quantified by densitometry. Collagen type I levels were elevated in the presence of nicotine (50 μg/ml). c Primary lung fibroblasts were treated with nicotine (50 μg/ml) for up to 72 h. Nicotine induced a significant increase in collagen type I mRNA levels at 48 h. d Fibroblasts were treated with nicotine (50 μg/ml) for up to 72 h. Protein bands were separated by SDS-PAGE gel electrophoresis (8%) and quantified by densitometry. Collagen type I protein was increased at 48 and 72 h by Western blot analysis. e Purified rat-tail collagen c was run with 72-h protein samples for antibody validation. Quantification of 1E included in 1D. All experiments repeated at least 3 times. Significance was assessed using p values <0.05 obtained by two-tailed t-test