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Fig. 1 | Respiratory Research

Fig. 1

From: Short-term intermittent cigarette smoke exposure enhances alveolar type 2 cell stemness via fatty acid oxidation

Fig. 1

Effect of short-term intermittent cigarette smoke (CS) exposure on alveolar stem cells. A Study design and experimental protocol. Adult Sftpc/GFP and C57BL6 mice were exposed to CS using this regimen. B–E Effect of different CS exposure protocols on AT2 stem cell function as determined with a colony-forming assay. Sorted EpCAMhigh/GFPhigh cells were cultured in an in vitro three-dimensional (3D) stem cell colony-forming assay. Representative day 14 images are presented for air-exposed (B), continuous CS-exposed (C), and intermittent CS-exposed alveolar colonies (D), demonstrating that colony-forming efficiency (CFE) of continuous CS-exposed AT2 cells was higher than that of air-exposed AT2 cells, and CFE of intermittent CS-exposed AT2 cells was higher than that of air-exposed and continuous CS-exposed AT2 cells. Quantification of CFE was conducted using triplicate wells (E). Data are presented as means ± SD. The reproducibility was confirmed from three independent experiments. *P < 0.05. Scale bars: 500 μm. F–I Whole lung sections from air-exposed and CS-exposed mice were immunohistochemically stained for Sftpc to identify AT2 cells. Sftpc-positive cells in the alveolar areas of air-exposed mice (F), continuous CS-exposed mice (G), and intermittent CS-exposed (H) mice were quantified for the percentage of Sftpc-positive cells from each group (I). Data are presented as means ± SD. N = 7–8 per group. *P < 0.05. Scale bars: 100 μm. J–L The proportion of AT2 cells within whole lung cells (WLCs) and epithelial cells. Dissociated WLCs were stained with EpCAM antibody and examined by flow cytometry. Representative flow cytometry dot plot for the WLCs of air-exposed (J), continuous CS- exposed (K), and intermittent CS-exposed (L) mice. M and N Percentage of GFPhigh (i.e., AT2) cells within WLCs (M) and EpCAMhigh (i.e., epithelial) cells (N) within the lungs of air-exposed, continuous CS-exposed, and intermittent CS-exposed mice. Data are presented as means ± SD. Data were compiled from three repeated experiments

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