cMUC5AC expression and MUC5AC-core genes in the small airway epithelium of healthy smokers. A. MUC5AC gene expression distribution in healthy smokers (blue, n = 60) compared to healthy nonsmokers (red, n = 72). Y-axis, number of subjects; X-axis, normalized MUC5AC expression. B. Volcano plot of MUC5AC-core genes comparing healthy smokers and healthy nonsmokers. Differentially expressed genes between smokers (n = 72) and nonsmokers (n = 60) were determined by unequal variance Student’s t-test followed by Benjamini-Hochberg correction (p < 0.05). Only MUC5AC-core genes were plotted. y-axis - negative log of p value; x-axis - log2-transformed fold-change; red dots – smoking up-regulated MUC5AC-core genes; blue dots – smoking down-regulated MUC5AC-core genes; grey dots – smoking unchanged MUC5AC-core genes. Numbers of each group are shown. C. Index of expression of the MUC5AC-core genes (% of abnormally expressed MUC5AC-core genes beyond that of healthy nonsmoker MUC5AC low-expressors in healthy nonsmokers (blue) compared to healthy smokers (red). Fold-change represents the average index difference between the groups, p value indicates significant differences between the groups. D. Unsupervised hierarchical clustering analysis of expression of MUC5AC-core genes in nonsmoker-high MUC5AC expressors (black, highest quartile of all healthy non-smokers, n = 15), nonsmoker-low MUC5AC expressors (green, lowest quartile of all healthy non-smokers, n = 15), smoker-high MUC5AC expressors (red, highest quartile of all healthy smokers, n = 18) and smoker-low MUC5AC expressors (blue, lowest quartile of all healthy smokers, n = 18). Genes expressed above the average are represented in red, below average in blue, and average in white.