(A) Decreased FOXJ1 expression in Smo and COPD AE, from early up to long-term ALI culture. Longitudinal analysis (middle graph) shows a strong, persistent downregulation in Smo and COPD, as compared with NS. At some time periods (as represented here in long-term ALI-AE), FOXJ1 expression correlated with the severity of the disease witnessed by the FEV1 (left graph). Correlation graph includes only Smo and COPD samples to specifically assess the correlation with the disease severity. $, £, and § highlight significant decreases in Smo, COPD1-2, and COPD3-4 patients, respectively, as compared with NS. (B) Decreased DNAI1 expression in Smo and COPD AE, from early up to long-term ALI culture. Longitudinal analysis (middle graph) shows a strong, persistent down-regulation in Smo and COPD, as compared with NS. At some time periods (as represented here in long-term ALI-AE), DNAI1 expression correlated with the severity of the disease witnessed by the FEV1 (left graph). Correlation graph includes only Smo and COPD samples to specifically assess the correlation with the disease severity. (C) Decreased numbers of bêta-tubulin IV-positive ciliated cells in Smo and COPD-derived ALI at every time-period. (D) Illustrative TSA-enhanced immunofluorescence staining for bêta-tubulin IV, demonstrating the differentiated emergence of ciliated cells in NS and COPD-derived ALI cultures. Scale bar, 50 μm. (E) Quantification of bêta-tubulin IV + surface in bronchial sections demonstrating reduced in situ expression of bêta-tubulin IV in native Smo and COPD AE, as illustrated by TSA-enhanced immunofluorescence staining for bêta-tubulin IV (right picture; scale bar, 50 μm). *, **, ***, **** indicate P-values of less than 0.05, 0.01, 0.001, and 0.0001, respectively. Bars indicate median ± interquartile range (A, B, E) or mean ± SD (C). AE, airway epithelium; ALI, air-liquid interface; COPD, chronic obstructive pulmonary disease; CT, control; FEV1, forced expired volume in 1 s; HKG, housekeeping genes; NS, non-smokers; SEM, standard error of the mean; Smo, smokers; y, years.