All imaging data were analyzed using ImageJ Fiji software. For each image, cells were counted relative to the measured length of the epithelium in micrometers (cells per micrometer). Mean cell counts per micrometer (cells per millimeter) were then calculated for never smokers (treated as the control), and individual values for each image from never and current smokers were calculated relative to the never smoker mean (i.e., relative cells per millimeter). We analyzed three images for each donor and assessed smoking-associated changes using the Wilcoxon rank-sum test. For panels in which MUC5AC was stained, current smoker tissue was assigned the phenotypic status of either MN or GCH based on qualitative assessment of goblet cell density and stratification. For each current smoker, three images of each status were analyzed.

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