Figure 8.

Differentiation of human bronchial epithelial cells in air-liquid interface culture is associated with an increase in Foxj1, Dnai2 and β-tubulin IV. Undifferentiated bronchial epithelial cells from monolayer cultures were plated on porous transwell inserts and differentiated in an air-liquid interface culture for 28 days. (A) Immunofluorescence detection of Foxj1, Dnai2 and β-tubulin IV in sections prepared at days 7, 14 and 28 after initiation of the air-liquid interface culture. Green staining (FITC) indicates the presence of Foxj1 or Dnai2, red staining (rhodamine) reflects β-tubulin IV. Blue staining indicates cell nuclei (DAPI). Scale bar 30 µm. Representative sections are shown and no detectable staining was observed for corresponding isotype controls. In (B) and (C), Western blots (B) and relative mRNA expression levels (C) for Foxj1, Dnai2 or β tubulin IV at day 7, 14 and 28 following initiation of the air-liquid interface culture are shown. Western blots are a representative of three independent experiments. The mRNA expression levels were quantified by real-time RT-PCR and evaluated as the x-fold change versus control (immediately before initiation of the air-liquid interface culture) using the 2^−ΔΔCt formula with GAPDH as endogenous standard. Results from mRNA analyses represent the means ± SEM of cultures from three donors and three experiments per donor. *P < 0.05 versus control.