Fig. 6.

TGF-β1 affects the composition of airway epithelium by decreasing the number of secretory epithelial cells in PBECs. PBEC were differentiated at the ALI followed by stimulation for 24 h with TGF-β1 in the presence or absence of 1,25(OH)₂D₃ or medium alone (CNTRL) to assess the mRNA expression of markers related to ciliogenesis (FOXJ1), club cells (SCGB1A1) and goblet cells (CLCA1) by qPCR (a). In addition, cells were stimulated for 48 h to assess the numbers of ciliated, club and goblet cells by confocal immunofluorescence (b). a Relative mRNA expression of FOXJ1, SCGB1A1 and CLCA1 was determined by qPCR. Normalized gene expression was calculated by using the expression of YWHAZ and RPL27 as reference genes. Fold changes in gene expression of the stimuli compared to CNTRL were first calculated, followed by a log-transformation of the data. Data are presented as individual values, including means ± SEM and were tested for significance using the two-way ANOVA and the Bonferroni post-hoc test (n = 6 donors). b Confocal immunofluorescence staining of ciliated (α-tubulin), club (CC-16), goblet (MUC5AC) and basal cells (P63) in ALI-PBEC (of 1 donor, which was confirmed in 3 other donors), DAPI (blue) was used to stain the nuclei and antibodies (Table 2) were used for the detection of luminal cell markers (green) and basal cells (P63, red), respectively. ** p < 0.01, *** p < 0.001. YWHAZ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta; RPL27, ribosomal protein L27.