Fig. 2.

HH pathway is activated during AEC differentiation. A, Curves representing the relative mRNAs levels normalized to GAPDH obtained during the course of ALI cultures by RT-qPCR (n = 5) for differentiation markers (CK5, non-differentiated cells; FOXJ1, ciliated cells; and MUC5AC/MUC5B, mucous-secreting cells) and HH pathway elements (GLI2/3; SMO and PTCH1). Means ±SEM of 2^−ΔΔCt are shown for each ALI time point. B, Representative immunoblots of total proteins extracted from ALI AEC cultures from ALI-7 to ALI-35 for the differentiation markers (Ck5 and Foxj1), HH pathway elements (Gli1/2/3; Smo; Ptch1) and Gapdh (n = 3). C, Histograms showing the relative increased expressions of Foxj1 and Gli2 protein levels as evaluated by western blot analysis after processing on ImageJ (Gapdh normalized ratios) during the course of AEC differentiation (in arbitrary units, AU). Results show mean ±SEM, *p < 0.05, **p < 0.01, ***p < 0.001. D, Representative confocal acquisitions from AEC cultures at ALI-14 and ALI-28 for the core HH pathway components Gli1, Gli2, Gli3, Ptch1 and Smo (all green); cilia (acetylated tubulin, red) and cell nuclei (DAPI, blue). Merged z-projections are shown with a magnification corresponding to the selected area.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)