Fig. 3: KRT5⁺ BC migration is directly modulated by ECM proteins.

a Flow cytometry confirmed expression of airway BC markers KRT5 and p63 in primary airway epithelial cells in submerged culture (passage 3). b Representative Airyscan maximum intensity projection image of KRT5⁺ BC demonstrating normal BC morphology and cytoskeletal arrangement on collagen I (n = 1 healthy control, n = 1 IPF patient); F-actin phalloidin stain (green) and DAPI (blue). Scale bar, 10 μm. c Normal capacity of BCs to undergo mucociliary differentiation shown here with representative H&E (c, upper) and PAS (c, lower) stained sections of agarose embedded air-liquid interface (ALI) cultures (n = 1 healthy control, n = 1 IPF patient, in duplicate). d Schematic of cell migration experimental design. Created with BioRender.com. e Individual cell track trajectories from all healthy (e, upper panels) and IPF (e, lower panels) donors plotted according to ECM ligand. Scale bars, 100 μm. f mean square displacement (MSD) of all KRT5⁺ BC tracks per ECM ligand (mean ± SEM). g–i Beeswarm SuperPlots showing migration data for KRT5⁺ BCs from healthy controls (n = 5) and patients with IPF (n = 5) cultured on collagen I (grey), collagen III (magenta), collagen IV (green) and versican (purple); g displacement over 12 h (healthy on collagen I vs. versican, P value = 0.0093; healthy on collagen III vs. versican, P value = 0.0005; healthy on collagen III vs. collagen IV, P value = 0.0040; IPF on collagen I vs. versican, P value = 0.001; IPF on collagen I vs. collagen IV, P value = 0.0014; IPF on collagen III vs. versican, P value < 0.0001; IPF on collagen III vs. collagen IV, P value < 0.0001). h mean track speed (healthy on collagen I vs. versican, P value = 0.0132; healthy on collagen I vs. collagen IV, P value = 0.0164; healthy on collagen III vs. versican, P value = 0.0049; healthy on collagen III vs. collagen IV, P value = 0.0061; IPF on collagen III vs. collagen IV, P value = 0.0122) and i straightness ratio (healthy on collagen I vs. versican, P value = 0.0007; healthy on collagen I vs. collagen IV, P value = 0.0083; healthy on collagen III vs. versican, P value = 0.0001; healthy on collagen III vs. collagen IV, P value = 0.0012; IPF on collagen I vs. versican, P value < 0.0001; IPF on collagen I vs. collagen IV, P value < 0.0001, IPF on collagen III vs. versican, P value < 0.0001, IPF on collagen III vs. collagen IV, P value < 0.0001). Each small dot (healthy) or square (IPF) represents cell-level data which is colour coded according to biological replicate. The larger circles (healthy) and squares (IPF) 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, ****P < 0.0001, **P < 0.01, *P < 0.05. Source data are provided as a Source Data file.