Normalized expression levels of the fat-depleting gene AZGP1 in large airway epithelium of 23 healthy nonsmokers and 36 healthy smokers. Ordinate: normalized gene expression levels for AZGP1 ± SEM. Shown is the data for the HG-U133 Plus 2.0 Gene microarray.

Normalized gene expression levels of AZGP1 from GEO deposited data of Spira et al⁴⁵ of the large airway epithelium with the HG-U133A chip ( http://www.ncbi.nlm.nih.gov/geo , accession number GSE994 ). Error estimates ± SEM.

Confirmation of the microarray results of AZGP1 expression levels in the large airway epithelium of healthy nonsmokers and healthy smokers with TaqMan real-time RT-PCR for AZGP1. The data included large airway epithelium from 17 healthy nonsmokers and 15 healthy smokers. Ordinate: expression level normalized to mean for all nonsmokers ± SEM.

Western analysis of AZGP1 protein expression in large airway epithelial cells of healthy nonsmokers and healthy smokers. Top, A (upper panel): AZGP1 protein expression in nonsmokers (lanes 1 to 3) and smokers (lanes 4 to 6). Same gel probed with anti β-actin antibody, a control for protein loading. Bottom, B: quantification by densitometry of AZGP1 to β-actin. The AZGP1/β-actin ratio is represented on the ordinate for the nonsmoker (top, A, upper panel) and smoker bands (top, A). Error bars represent the SE.

Immunohistochemistry assessment of AZGP1 expression (shown in red) in endobronchial biopsy specimens from a healthy nonsmoker and a healthy smoker. Top left, A: endobronchial biopsy specimen from a healthy nonsmoker stained with rabbit IgG1 isotype control. Top center, B: endobronchial biopsy specimen from a healthy nonsmoker, stained with rabbit antihuman AZGP1 antibody. Top right, C: endobronchial biopsy specimen from a healthy nonsmoker stained with rabbit antihuman AZGP1 antibody. Bottom left, D: endobronchial biopsy specimen from a healthy smoker stained with rabbit IgG1 isotype control. Bottom center, E: endobronchial biopsy specimen from a healthy smoker stained with rabbit anti-human AZGP1 antibody. Bottom right, F: endobronchial biopsy specimen from a healthy smoker stained with rabbit antihuman AZGP1 antibody. Bar = 0.01 mm. Original magnification for all panels is ×40.

Immunofluorescence assessment of AZGP1 colocalization with mucin 5AC and chromogranin A. Cytospin preparations of large airway epithelium from a healthy nonsmoker were stained with antibodies against AZGP1, mucin 5AC, and chromogranin A, followed by a Cy5-conjugated secondary antibody for AZGP1 (shown in green) and a Cy3-conjugated antibody for mucin 5AC and chromogranin A (shown in red). Nuclei were stained with DAPI (shown in blue); colocalization of AZGP1 with either mucin 5AC or chromogranin A is shown in yellow. Colocalization of AZGP1 and mucin 5AC (panels A to I). Phase contrast image of a cytospin preparation from a healthy nonsmoker showing large airway epithelial cells (top left, A). Top center, B: cytospin preparation from a healthy nonsmoker (same field as in top left, A), but in the Cy5 channel showing AZGP1 staining of large airway epithelial cells. Top left, C: cytospin preparation from a healthy nonsmoker (same field as in top left, A), but in the Cy3 channel showing mucin 5AC staining. Top middle left, D: cytospin preparation from a healthy nonsmoker (same field as in top left, A) showing colocalization of AZGP1 and mucin 5AC (shown in yellow); nuclei are shown in blue. Top middle center, E: cytospin preparation from a healthy nonsmoker (same field as in top left, A) viewed in phase contrast showing colocalization of AZGP1 and mucin 5AC as well as DAPI nuclear staining. Top middle right, F: cytospin preparation from a healthy nonsmoker (same field as in top left, A) demonstrating very minimal to no autofluorescence of large airway epithelial cells. Middle left, G: cytospin preparation from a healthy nonsmoker viewed in phase contrast showing a secretory airway epithelial cell. Middle center, H: cytospin preparation from a healthy nonsmoker (same field as in middle left, G) viewed with the Cy3 and Cy5 channels (stained with both AZGP1 and mucin 5AC antibodies), showing a secretory airway epithelial cell positive for mucin 5AC but negative for AZGP1; nuclei are shown in blue. Middle right, I: cytospin preparation from a healthy nonsmoker (same field as in middle left, G) showing an example of AZGP1-negative mucin 5AC-positive secretory airway epithelial cell shown as three-channel fluorescent view (including phase view). Colocalization of AZGP1 and chromogranin A (panels J–O). Bottom middle left, J: phase contrast view of cytospin preparation from a healthy nonsmoker showing airway epithelial cell. Bottom middle center, K: cytospin preparation from a healthy nonsmoker (same field as in bottom middle left, J) showing an example of colocalization AZGP1 and chromogranin A in an airway epithelial cell; nucleus is shown in blue. Bottom middle right, L: cytospin preparation from a healthy nonsmoker (same field as in bottom middle left, J) showing an example of colocalization of AZGP1 and chromogranin A in an airway epithelial cell shown as three-channel fluorescent view (including phase view). Bottom left, M: phase contrast view of cytospin preparation from a healthy nonsmoker showing airway epithelial cell. Bottom center, N: cytospin preparation from a healthy nonsmoker (same field as in bottom left, M) showing an example of colocalization AZGP1 and chromogranin A in an airway epithelial cell; nucleus is shown in blue. Bottom right, O: cytospin preparation from a healthy nonsmoker (same field as in bottom left, M) showing an example of colocalization of AZGP1 and chromogranin A in an airway epithelial cell shown as three-channel fluorescent view (including phase view). Bar = 0.01 mm. Original magnification for all panels is ×60.

Quantification of AZGP1-positive cells in healthy nonsmokers and healthy smokers. Cytospin preparations of large airway epithelial cells from five nonsmokers and six smokers were stained with rabbit antihuman AZGP1 or rabbit IgG as control. Goat antirabbit Cy3 was used as a secondary antibody. Nuclei were counterstained with DAPI. The percentage of AZGP1-positive cells was calculated as the number of AZGP1-positive cells per field/total number of nuclei per field in 10 random ×60 fields for each subject. Representative low-power (original ×20) staining of specimens from one healthy nonsmoker and one healthy smoker are shown. Top left, A: healthy nonsmoker, rabbit IgG1 isotype control. Top center, B: healthy smoker, rabbit IgG1 isotype control. Bottom left, C: healthy nonsmoker, rabbit antihuman AZGP1 antibody. Bottom center, D: healthy smoker, rabbit antihuman AZGP1 antibody. Bar = 0.01 mm. Right, E: quantification of AZGP1-positive cells; the ordinate shows percentage of AZGP1-positive cells; the abscissa shows the different groups (n = 5 healthy nonsmokers, n = 6 healthy smokers). Error bars represent the SD.