Fig. 4: Interaction with a 3D cell-derived matrix induces gene expression changes in migratory KRT5⁺ BCs.

a Schematic of experimental design for KRT5⁺ BC migration on CDM. Created with BioRender.com. b Images showing generation of CDM from primary HLFs (n = 1, in duplicate) b,i pre- and b,ii-iii post- decellularization; F-actin (phalloidin, green), DAPI (blue), collagen I (red) and fibronectin (purple). c Individual cell track trajectories from healthy n = 4 (black) or IPF n = 4 (red) KRT5⁺ BCs plotted from centroid; Scale bar, 100 μm. d Mean square displacement of all healthy KRT5⁺ BCs (black) vs. IPF KRT5⁺ BCs (red) on IPF CDMs (mean ± SEM). Beeswarm SuperPlot showing e displacement over 12 h, f mean track speed, and g straightness ratio for KRT5⁺ cells from healthy controls (n = 4, ≥2 replicates per donor) and IPF patients (n = 4, ≥2 replicates per donor) tracked over IPF HLF CDM. Each small dot represents cell-level data which is colour coded according to biological replicate. The larger circles represent the mean value per biological replicate. Summary statistics with mean and standard deviation are superimposed on the plot. Sample-level means compared using a one-way ANOVA with Tukey’s multiple comparison test. h Schematic of experimental design to assess gene expression changes induced by culture of healthy (n = 3) or IPF (n = 3) KRT5⁺ BCs on collagen I (2D) vs. IPF CDM (3D). i Principal component analysis of gene array data; unit variance scaling was applied to rows; SVD with imputation was used to calculate principal components. Prediction ellipses are such that with probability 0.95, a new observation from the same group will fall inside the ellipse. j Heatmap with hierarchical clustering of normalised gene expression. Rows and columns using correlation distance and average linkage. Rows are centred and unit variance scaling is applied. k Volcano plots showing gene expression changes when healthy (left) or IPF (right) KRT5⁺ BCs were cultured on IPF CDM vs. collagen I. Upregulated genes (red), downregulated genes (blue). Significance defined as log fold change >2 or <2, P < 0.05. P values were calculated based on a Student’s t test of the replicate normalised gene expression values (2^(- Delta CT)) for each gene in control and test groups. The P value calculation was based on parametric, unpaired, two-sample equal variance, two-tailed distribution. Housekeeping genes used - ACTB, B2M and RPLP0. Source data are provided as a Source Data file.