Figure 4. Overexpression of ProT increases protein acetylation in emphysematous lungs of mice and patients.

(a) Immunohistochemistry (left) and quantitation (right) of acetylated-lysine (acetyl-K) in lung tissues of 4-day-old ProT HZ, HET and NT mice. (b) A positive correlation (P=0.0012; Pearson’s correlation coefficient) between the levels of ProT immunoreactivity and those of acetylated-lysine immunoreactivity. (c) Immunohistochemistry (upper) and quantitation (lower) of acetyl-K in lung tissues from patients with emphysema of varying severity and non-COPD individuals. Values shown are immunoreactive levels in individual specimens; horizontal bars represent the mean. (d) Immunofluorescence microscopy. Lung tissues from patients with severe emphysema and from non-COPD individuals were incubated with mouse monoclonal antibody against ProT and rabbit polyclonal antibody against acetyl-K followed by Alexa Fluor 594-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG. Nuclei were counterstained with DAPI. Fluorescence signals for ProT (red), acetyl-K (green) and the nucleus (blue) were examined by fluorescence microscopy. (e) A positive correlation (P<0.0001; Pearson’s correlation coefficient) between the levels of ProT immunoreactivity and those of acetyl-K immunoreactivity. (f,g) Immunohistochemistry (left) and quantitation (right) of acetyl-K in lung tissues from ProT HET and NT mice that were injected intraperitoneally with CSE or saline twice a week for 6 weeks (f) and from FVB mice that had been intratracheally injected with lentiviral vectors expressing ProT or luciferase (Luc) shRNA followed by intraperitoneal injections of CSE or saline twice a week for 6 weeks (g). Acetyl-K immunoreactive intensity (mean±s.e.m.; Student’s t-test) was analysed in three randomly selected fields at × 400 magnification in each section by MetaMorph software (n=3 in (a,f,g); n=4–8 in c). Values shown are relative acetyl-K intensity levels, with the level in normal tissues arbitrarily set to 100. Scale bar=20 μm, and the boxed areas in upper panels (original magnification × 400) are magnified and shown in lower panels. Results are representative of three independent experiments.