Figure 1

Primary human airway epithelial basal/progenitor cell cultures. A. Immunocytochemical verification of basal cell phenotype. Airway basal cells from healthy nonsmokers (n = 14) were purified by culturing large airway epithelial cells obtained by bronchoscopy and brushing. After 7 days of culture, the cells were trypsinized and cytospin preparations were assessed for expression of cytokeratin 5 (KRT5; basal cell-specific marker); β-tubulin IV (TUBB4; marker of ciliated cells); mucin 5AC (MUC5AC; secretory cell marker); and N-cadherin (CDH2; marker for mesenchymal cells). B. TaqMan quantitative real-time RT-PCR assessment of mRNA expression of the basal cell markers KRT5, KRT14 and tumor protein p63 (TP63) in complete large airway epithelial cell population (n = 5 subjects) recovered by airway brushing (blue bars) compared to basal cell cultures (day 7; red bars. n = 3 subjects). C. Western analysis for the basal cell markers KRT5 and KRT14, the cilia markers dynein, axonemal, intermediate chain 1 (DNAI1) and acetylated α-tubulin (Ac-TUBA), the secretory cell marker secretoglobin family 1A member 1 (SCGB1A1) and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Lane 1 – differentiated airway epithelium obtained by brushing. Lane 2 - cell extracts from basal cell cultures (day 7). D. Forkhead box J1 (FOXJ1), regulatory factor X3 (RFX3), Not homeobox (NOTO) and GAPDH mRNA expression during basal cell differentiation on air liquid interface (ALI) over 28 days (n = 3 subjects per time point). The expression was assessed by TaqMan quantitative real-time RT-PCR and a gene was deemed “undetectable” if not detected after 40 cycles of assessed undiluted cDNA. B = basal cells not cultured on ALI. * p < 0.05; ** p < 0.01; *** p < 0.005 compare all to GAPDH.