Figure 4. Cell cycle arrests during signaling-induced loss of airway epithelial mucociliary differentiation, but is not required for differentiation.

(A) Fold change in cell cycle transcriptional program usage (defined in Fig. S5A) predicts a reduction in cell cycle in response to several signaling conditions.

(B) Expression of cell cycle inhibitor genes in control, BMP4, CHIR, IFNG and TGFB1 induced cells.

(C) Representative immunofluorescence images of whole-mount undifferentiated hBEC cultures (300–400 individual cells per image) treated with indicated cytokines and stained for DNA (Hoechst; white) indicate reduced cell densities in conditions showing reduced cell cycle programs and increased cell cycle inhibitor gene expression. Scale bars, 50 μm.

(D) Quantification of areal cell density from panel C. Bars represent mean ± SEM, n = 3 HBEC donors (BMP4: n=2), calculated from 1 mm stitched images. *p≤0.05 by Wilcoxon rank-sum test.

(E,F) Fraction of cells incorporating EdU after 48 hours of continuous EdU incubation (E) 48-hours following epithelial stripping and treatment with the indicated cytokines, or (F) 48-hours following epithelial stripping and treatment with aphidicolin (2 μg/mL), PD0332991 (PD033, 100 nM), or thymidine (thy.) block (2 mM). Bars represent mean ± SEM, n=3 HBEC donors.

(G) Top: Representative images for immunofluorescence of whole mount differentiated hBEC cultures (approximately 1000 individual cells per image) treated with aphidicolin, thymidine block, and PD0332991 and stained for MUC5B (red) and FOXJ1 (green). Bottom: Quantification of images to calculate percent MUC5B and FOXJ1 cells. Scale bars, 100 μm.

(H) Fold change of mRNA expression in differentiated HBEC cultures treated with aphidicolin/PD0332991 over untreated differentiated HBECs. Axis in logarithmic scale. Bars represent mean ± SEM, n=3 HBEC donors.