Comparison of the differentiation capacity of immortalized BCi-NS1 cells to that of primary basal cells. Primary airway BC and immortalized BCi-NS1 cells were cultured under differentiation inducing conditions on air-liquid interface (ALI). A, B. mRNA transcripts; A. Primary BC. B. BCi-NS1.1 cells. At ALI day 0 and day 28, TaqMan quantitative PCR analysis was used to assess cell type specific mRNA markers: KRT5 (basal cell); TP63 (basal cell); MUC5AC (secretory cell); MUC5B (secretory cell); TFF3 (goblet cell); CC10 (Clara cell); DNAI1 (ciliated cell) and FOXJ1 (ciliated cell). Data shown is the average ± the standard error of n = 5 independent experiments for primary BC and n = 10 independent experiments for BC-NS1.1 cells. Statistics were calculated by a 2-tailed Student’s t test. Asterisk (*) indicates not detected. For B, the data represents an average ± the standard error for BCi-NS1.1 cells from passage 6 to 43. C. Characterization of differentiation by Western analysis of cell type specific markers. Lane 1 – Primary BC, ALI day 0; lane 2 – Primary BC, ALI day 28; lane 3 – BCi-NS1.1, ALI day 0; and lane 4 – BCi-NS1.1, ALI day 28. Shown is the expression of a basal cell marker (TP63); Clara cell marker (CC10); and ciliated cell markers (DNAI1 and FOXJ1). GAPDH was used as a loading control. D, E, F. Immunofluorescent staining of ALI day 28 primary BC and BCi-NS1.1 cells for D. CC10 (Clara cell, red), E. MUC5AC (secretory cell, green) or F. TFF3 (goblet cell, red) and nuclei (blue). Bar = 20 μm. For C-F all data shown is representative of n = 3 independent experiments.