Fig. 6: Smoking inhibits the early ciliating state.

a Wholemount IF labeling of FOXN4 KO in human tracheal ALI cultures. Arrows, immature (white) and mature ciliated cells (yellow). Scale bar = 25 μm. b Sample axonemal phenotypes by acetylated α-Tubulin IF labeling (white). Arrows, FOXN4 KO cells displaying absent (white), short or sparse (yellow), or bulging (orange) axonemes were classified immature; dense, well-formed axonemes (blue) were classified mature. Scale bar = 25 μm. c Quantified fraction of mature and immature ciliated cells from control (n = 584) and FOXN4 KO cultures (n = 854) in b. Bar plots: mean fractions ± standard error (n = 5 confocal fields); points: fraction for each field. d Wholemount IF labeling of FOXN4 KO illustrates basal bodies are generated (γ-Tubulin, green), but fail to dock (white arrows) and deuterosomes are assembled (DEUP1, red), but retained (yellow arrows). Scale bar = 25 μm. e UMAP with subclustering of in vivo ciliated cells. Mature ciliated cells combine two subgroups (Supplementary Fig. 8b). f Average expression of markers from three in vitro ciliogenesis states and in vivo mature mucus secretory cells (far right). Box centers: median; upper/lower box bounds: first/third quartiles; upper/lower whiskers: extend from upper/lower bounds up to/down from the largest/smallest value, no further than 1.5× the inter-quartile range from upper/lower bounds; points: outliers. N (left to right) = 140; 2216; 30,866; 3026 cells. g (Left) Hybrid secretory/ciliated cells exhibit MUC5B labeling and early ciliogenesis phenotypes via TUBG1 morphology under confocal IF microscopy⁸² (day 16 ALI cultures imaged 48 h post-DAPT). Scale bar = 10 μm. (Right) TUBG1 pattern schematic for non-ciliated, early ciliating, and mature multiciliated cells. h Functional gene network (FGN) of non-core upregulated genes in mature ciliated cells. Edges: connect genes annotated for the same enrichment terms; node colors: functional metagroups containing genes (exemplar terms, right); white nodes: hub genes (in multiple metagroups); node size: gene connectivity; label size: mean log fold-change between smokers and non-smokers; edge thickness: number of shared terms; edge color: metagroup membership of the connected node(s) if one or both are not hub genes; gray edges connect two hub genes. i FGN for genes downregulated by smoking in hybrid secretory/ciliated cells (network as described in h). See Supplementary Fig. 8.