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. 2004 Nov 1;114(9):1248–1259. doi: 10.1172/JCI21146

Figure 2.

Figure 2

Cigarette smoke exposure causes lung cell apoptosis as assessed by TUNEL in Nrf2–/– lungs. (A) Lung sections (n = 5 per group) of room air– or CS-exposed (6 months) Nrf2+/+ or Nrf2–/– mice were subjected to TUNEL (left column) and DAPI stain (middle column). Merged images are shown in the right column. CS-exposed Nrf2–/– mice show abundant TUNEL-positive cells (arrows) in the alveolar septa. Magnification, ×20. (B) Quantification of TUNEL-positive cells (per 1,000 DAPI-stained cells). The number of TUNEL-positive cells was significantly higher in the CS-exposed Nrf2–/– mice as compared with their wild-type counterparts (*P – 0.05). Values represent mean ± SEM. (C) Identification of apoptotic (TUNEL-positive) type II epithelial cells (left column), endothelial cells (middle column), and alveolar macrophages (right column) in the lungs of CS-exposed (6 months) Nrf2+/+ and Nrf2–/– mice. Type II epithelial cells, endothelial cells, and alveolar macrophages were detected with anti-SpC, anti-CD34, and anti–Mac-3 antibodies, respectively, as outlined in Methods. Nuclei were detected with DAPI (blue). Shown are the merged images, with colocalization (yellow arrows) of cell-specific markers (cytoplasmic red signal) and apoptosis (nuclear green + blue DAPI signal, resulting in a lavender-like signal); non-apoptotic (TUNEL-negative) cells with positive cell specific marker (red signal) are highlighted with a red arrow. TUNEL-positive apoptotic cells lacking a cell-specific marker are highlighted by white arrowheads. The majority of TUNEL-positive cells consisted of endothelial and type II epithelial cells, whereas most alveolar macrophages were TUNEL negative. Scale bars: 5 μm.