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. Author manuscript; available in PMC: 2025 Apr 17.
Published in final edited form as: Cell Syst. 2024 Mar 19;15(4):307–321.e10. doi: 10.1016/j.cels.2024.02.005

Figure 3. Evidence of convergent and pathway-specific transcriptional responses including loss of canonical cell identity.

Figure 3.

(A) Number of genes showing >2-fold differential expression in basal, secretory (club, goblet), multiciliated and rare (ionocyte, tuft, PNEC) cells following each treatment. Empty boxes = no genes; N/A = no cells present.

(B) Schematic for gene program analysis of signaling responses.

(C) Mean usage of eleven treatment-induced gene programs across all cells from each treatment condition the presence of convergent (Shared-1–3) and pathway-specific programs (remaining programs). A further nine control programs are shown in Fig. S5B. The heatmap is first column-normalized (sum=1) and then row-normalized (max= 1).

(D) Transcriptional responses vary across cell types as seen from the mean usage of signaling programs. For shared programs, usage is averaged across all treatments; for perturbation-specific programs, usage is calculated for cells from one perturbation.

(E) Loadings of top 10 genes for each of the signaling induced program (left) and of epithelial KRT genes (right) across all signaling programs; full table for gene loadings is provided in Table S6.

(F) Putative loss of canonical basal cell identity, but not mucociliary cell identities, is observed by the near-complete replacement of control transcriptomic programs (grey) in basal cells by induced programs (red) in response to CHIR, IFNG, and TGFB1. Other treatments are shown for contrast.

(G) Representative immunofluorescence images (300–400 individual cells per image) of whole-mount differentiated HBEC cultures treated with indicated cytokines and stained for F-actin (white), acetylated-alpha-tubulin (red), and DNA (Hoechst; blue). Scale bars, 50 μm. Bottom row conditions, corresponding to loss of basal cell identity in panel F, show cytoskeletal disorganization.

(H) Quantification of epithelial permeability measured by transit of lucifer yellow dye across the epithelial surface of cultures treated with indicated cytokines. Bars represent mean ± SEM, n = 3 HBEC donors (IFNG: n=2; IFNA: n=1) biological replicates shown as points, normalized to untreated fluorescence = 1. * = p-value ≤0.05 by Wilcoxon rank sum test.