Extended Data Fig. 4: mTECs cease proliferating during differentiation.
From: An alternative cell cycle coordinates multiciliated cell differentiation
a, Representative immunofluorescence images of wild-type mTECs cultured at air-liquid interface for five days and stained for EdU (cyan), FOXJ1 (red) and nuclei (Hoechst, grey). EdU or DMSO was added during days one to five of culture at air-liquid interface. n = 3 biological replicates. Scale bar, 10 μm. b, Percentage of multiciliated cells (expressing FOXJ1) that exhibit EdU incorporation. Bar graph quantitates EdU incorporation in 186 multiciliated cells assessed from 3 biological replicates. c, Immunofluorescence images of differentiating wild-type mTECs. mTECs were stained for Histone 3 phosphorylated at serine 10 (H3S10P, cyan), TP63 (yellow) and nuclei (Hoechst, grey) two days before transition to air-liquid interface (Day -2), one day before (Day -1), the day of transition (Day 0), one day after transition to air-liquid interface (Day 1) or two days after (Day 2). H3S10P is a marker of cells in mitosis. TP63, also known as p63, is a marker of airway stem cells. Lower panels depict individual channels. Scale bar, 10 μm. d, Percentage of H3S10P-expressing cells in differentiating mTECs at the indicated times. Horizontal lines indicate means ± s.e.m. of 3 biological replicates, ****P = 0.000002 and NS, not significant (one-way ANOVA with Sidak’s correction). e, Percentage of TP63-expressing airway stem cells that also express H3S10P in differentiating mTECs at the indicated times. Horizontal lines indicate means ± s.e.m. of 3 biological replicates, ***P = 0.0009 and NS, not significant (one-way ANOVA with Sidak’s correction).
