(A–F) BCIs in ALI culture for up to 4 weeks. Human recombinant DKK1 (DKK1) was used to inhibit Wnt/β-catenin signaling starting at ALI day 7 (D7).
(A) Confocal imaging of specimens stained for Ac.-α-tubulin (MCCs, blue), CC10 (Club cells, green), and Actin (cell membranes, magenta) shows moderately increased MCC and Club cell numbers after DKK1 treatment.
(B) DKK1 leads to a transient decrease in BCs but does not lead to loss of ΔN-TP63+ (green) cells. Nuclei (DAPI, blue).
(C) Quantification from (A) and (B), respectively. Mann Whitney test, not significant, ns (p > 0.05); ***p ≤ 0.001.
(D) Quantitative real-time PCR expression levels are depicted relative to stage controls. DKK1 increases expression of MCC (FOXJ1, MCIDAS) and to a lesser extent Club cell (SCGB1A1) markers but without reduction of BC markers (ΔN-TP63, KRT5). Student’s t test, not significant, ns (p > 0.05); *p ≤ 0.05; **p ≤ 0.01.
(E and F) Optical orthogonal sections of confocal images after staining for BC markers ΔN-TP63 (green, in E) and KRT5 (green, in F).
Related to Figure S5.