Figure 2. Distinct modes of basal and secretory cell metaplasia in response to different signals.

(A) UMAP of all scRNA-Seq data from Fig. 1D (this and panel B use data GEO #GSE246368, n=3 donors per condition, 2 donors for FGF2, and 1 donor for IFNG, OSM), colored by canonical cell-type annotations learnt from untreated controls.

(B) Gene expression heatmap showing that the classified cells across treatments preserve expression of marker genes for their respective cell types. Each row represents a single meta-cell showing average expression of 10 nearest neighbors; classified cell types on left.

(C) Frequency of cell types after perturbation (logarithmic scale). Top: dynamic range of signaling-induced changes. This and panels (D-F, H-J,L) incorporate data (GEO # GSE246441, 3 additional donor replicates for OSM, IFNG, and control analyzed by 10X). Red=maximum; Blue=minimum; Black=untreated baseline. Bottom: heatmap of donor-averaged cell type frequencies in all conditions.

(D,E) First two Principal Components of the cell type frequency matrix, after per-donor normalization, showing (D) values for each treatment, and (E) cell type loadings. PC1 corresponds to basal cell metaplasia, and PC2 corresponds to goblet cell hyperplasia.

(F) Fold change in cell type frequencies for four conditions with highest PC1 values corresponding to loss of canonical differentiation. ND = Not detected

(G) Representative immunofluorescence images of cross-sections of differentiated HBEC cultures (15–50 individual cells represented per image) treated with indicated cytokines and stained for KRT5 (white), MUC5B (green), and acetylated alpha-tubulin (red). Scale bars, 25 μm.

(H) Log-Fold change goblet cell abundance for the three conditions with >2-fold increase in goblet cell frequency. Points = donors; bar = mean.

(I) Comparison of changes in frequency of goblet and club cells. Conditions inducing goblet cell hyperplasia are highlighted (black); remaining conditions shown in gray. See also Fig. S4B. Both axes in (I,J) are in logarithmic scale.

(J) Comparison of changes in frequency of goblet and multiciliated cells in the context of goblet cell hyperplasia. Colors as in (I). See also Fig. S4C.

(K) Gene expression heatmap showing differences in goblet cell states induced by IL13 and IL17. On the left we have single goblet cells where each column is a single meta-cell as in panel B. On the right we have plotted average expression across all goblet cells in control, IL17A and IL13 conditions. CP10k = Counts per total 10k counts.

(L) Comparison of changes in frequency of ionocytes and PNEC+Tuft cells across all conditions, indicating tandem variation in the frequency of rare cell types. See also Fig. S4D.