A, Abundance and localization of FoxO3 in lung alveolar/airway epithelial cells of nonsmokers, smokers and patients with COPD. Red color represents the presence of FoxO3 (indicated with arrow), which was decreased in lungs of smokers and patients with COPD. (-) control = negative control which was stained without primary antibody Alv = alveoli; Aw = airway; E = epithelial cells. Original magnification, × 200. B, Immunostaining scores for FoxO3 per cell-type in alveolar and airway regions of the lung. The assessment of immunostaining intensity was performed semiquantitatively and in a blinded fashion. Immunoblot analysis of FoxO3 in whole tissue lysates extracted from the lung tissue (C) and sputum cells (E) of nonsmokers, smokers, and patients with COPD. After densitometric analysis, the values of FoxO3 in lung tissue (D) and sputum cells (F) were normalized against β-actin (loading control). The relative levels of FoxO3 were significantly decreased in lung tissues of smokers and patients with COPD compared to nonsmokers. G, Whole tissue lysates extracted from the lung tissues of nonsmokers, smokers, and patients with COPD were immunoprecipitated with anti-FoxO3 antibody, and immunoprecipitates were subjected to immunoblot and probed with anti-acetylated lysine antibody. H, Relative intensity of acetylated lysine/FoxO3 represents the increased acetylation of FoxO3 in lungs of smokers. Data are shown as mean ± SEM (n= 3 to 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared to nonsmokers. ⁺P < 0.05, significant compared with smokers.

A, Immunoblot analysis of FoxO3 in whole tissue lysates extracted from lungs of mouse exposed to CS for 3 days and 4 months. B, After densitometric analysis, the values of FoxO3 were normalized against β-actin (a loading control), respectively. C, Nuclear fraction from lungs of mouse exposed to CS for 3 days and 4 months were immunoprecipitated with anti-FoxO3 antibody, and immunoprecipitates were subjected to immunoblot analysis and probed with anti-acetyl-lysine antibody. IgG was used as an isotype control. D, The relative density of acetylated FoxO3 showed increased acetylation of FoxO3 in CS exposed mice lung at 3 days and 4 months. A representative blot is shown. Data are shown as mean ± SEM (n= 3 to 4 per group). ***P < 0.001, significant compared to air-exposed WT mice.

The pictures shown are H&E stained lung sections from air- or CS-exposed WT and FoxO3^-/- mice for 3 days (A), 8 weeks (B) and 4 months (C). Arrows indicate airspace enlargement. Original magnification is × 200. Mean Linear Intercept (Lm) was analyzed in H&E stained slides (D, 3 days; E, 8 weeks; F, 4 months). Lung sections from CS-exposed FoxO3^-/- mice for 4 months show an increased airspace enlargement when compared with CS-exposed WT mice. Data are shown as mean ± SEM (n=3 per group). ***P < 0.001, significant compared with corresponding air-exposed mice; ⁺⁺⁺P < 0.001, significant compared with CS-exposed WT mice.

A, Tissue resistance, static lung compliance and tissue elastance were measured by FlexiVent after 4 months of CS exposure. B, Mice exposed to air or CS for 4 months were subjected to running on the treadmill at 24 h after the last exposure. Running time and running distance were measured. Data are shown as mean ± SEM (n=4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with corresponding air-exposed mice; ⁺P < 0.05, ⁺⁺P < 0.01, significant compared with CS-exposed WT mice.

The number of total cells in BAL fluid from air- or CS-exposed mice for 3 days (A), 8 weeks (B) and 4 months (C) was determined. Lavaged neutrophil numbers were counted in Diff-Quik stained cytospin slides, which were prepared using BAL fluid. Quantification of neutrophils expressed as % of total cells in BAL fluid from air- or CS-exposed mice for 3 days (D), 8 weeks (E) and 4 months (F). Data are shown as mean ± SEM (n=3 to 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with corresponding air-exposed mice; ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with CS-exposed WT mice.

Mac-3 positive cells were identified by dark brown immunohistochemical staining, which were indicated by arrows in lung sections from air- or CS-exposed mice for 3 days (A), 8 weeks (B) and 4 months (C). The representative pictures shown are from at least three separate experiments. Quantification results are presented as the number of Mac-3-positive cells in lung sections from 3 days (D), 8 weeks (E) and 4 months (F) air- or CS-exposed mice. Original magnification, × 200. Data are shown as mean ± SEM (n=3 per group). ***P < 0.001, significant compared with corresponding air-exposed mice; ⁺P < 0.05, ⁺⁺P < 0.01, significant compared with CS-exposed WT mice.

The level of proinflammatory mediators, such as MCP-1, KC and IP-10 are measured in lung homogenates obtained from air- or CS-exposed WT and FoxO3^-/- mice for 3 days (A), 8 weeks (B) and 4 months (C). Data are shown as mean ± SEM (n= 3 to 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared to corresponding air-exposed mice. ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 significant compared with CS-exposed WT mice.

A, NF-κB-DNA binding was measured by the Trans-AM transcription factor RelA/p65 ELISA kit in nuclear proteins from CS-exposed mice lung for 3 days. Data are shown as mean ± SEM (n= 3 to 4 per group). *P < 0.05, **P < 0.01, significant compared to corresponding air-exposed mice. ⁺P < 0.05, significant compared with CS-exposed WT mice. B, Nuclear fraction from mouse lung exposed to CS for 3 days were immunoprecipitated (IP) with anti-RelA/p65 antibodies. C, Reciprocal IP of nuclear fraction from mouse lung exposed to CS 3 days was performed with anti-FoxO3 antibodies, followed by immunoblot analysis. D, H292 epithelial cells were treated with CSE (1%), and nuclear fractions from cell lysates were used for IP with anti-RelA/p65 antibody. IgG was used as an isotype control. Immunoprecipitates were subjected to immunoblot analysis and probed with anti-FoxO3 or anti-RelA/p65 antibody.

The levels of MnSOD and catalase were measured by RT-PCR (A) and immunoblot (B) at the indicated time points of CS exposures. CuZnSOD was used as a non-specific target protein of FoxO3, and GAPDH was used for loading controls. After densitometric analysis, the values of MnSOD and catalase were normalized against GAPDH, respectively. Gel pictures shown are representative of at least three separate experiments. Data are shown as mean ± SEM (n= 3 to 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared to corresponding air-exposed mice. ⁺⁺⁺P < 0.001, significant compared with CS-exposed WT mice.