Figure 6. Overexpression of ProT increases NF-κB acetylation in emphysematous lungs of mice and patients.

(a–c) Immunohistochemical detection of acetyl-NF-κB p65 in lung tissues from 4-day-old ProT HZ, HET and NT mice (a), from patients with emphysema of varying severity and non-COPD individuals (b) 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 (c). Note that positive nuclear staining for acetyl-NF-κB p65 in the lung epithelium of ProT transgenic mice was observed (a) and that higher levels of acetyl-NF-κB p65 accumulation were detected in patients with more severe emphysema (b). The boxed areas in upper panels (original magnification × 400; Scale bar=20 μm) are magnified and shown in lower panels. (d,e) Immunofluorescence microscopy. Lung tissues from ProT HET and NT mice that had been injected intraperitoneally with CSE or saline twice a week for 6 weeks (d) and mouse lung epithelial MLE 12 cells that had been transduced with ProT and GFP by lentiviral vectors Lenti-ProT and Lenti-GFP, respectively, and then treated with CSE or saline (e) were incubated with mouse monoclonal antibody against ProT and rabbit polyclonal antibody against NF-κB p65 or acetyl-NF-κB p65 (Lys310) followed by Alexa Fluor 594-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG. Nuclei were counterstained with DAPI. Fluorescence signals for ProT (red), NF-κB p65 (green), acetyl-NF-κB p65 (green) and the nucleus (blue) were examined by fluorescence microscopy. Scale bar=10 μm; original magnification × 400. Results are representative of three independent experiments.