Figure 5
Effect of RA on cell differentiation of BBEC cultures. BBEC cultures were grown for 21 days at an ALI with varying concentrations of RA before fixation. The BBEC cultures were subsequently immunostained to assess (A) ciliation (cilia - green; F-actin - red; nuclei - blue) and (B) mucus production (mucus - green; cilia - red; nuclei - blue) or (C) examined by SEM. Representative images are shown of BBECs grown in the presence of (i) 0 and (ii) 100 nM RA (see Figs. S5A, B and D). Quantitative analysis of ciliation of the apical surface of BBEC cultures grown in the presence of 0, 25, 50, 100 and 250 nM RA was performed using (D) fluorescence intensity thresholding of immunostained cultures (see Fig. S5A) and (E) by counting the number of ciliated cells per field of view in H&E-stained sections (see Fig. S4A) as described in Fig. 2. Statistical significance was tested using an Ordinary one-way ANOVA: ** = P < 0.01; **** = P < 0.0001.
