Recombinant TNF-α and IFN-γ alter differentiation of the tracheal epithelial cell in vitro. A and B: images showing the frequency of basal (Trp63+), ciliated (Tubb4b+), and club (Pb1fa1+) cells fed the control medium. C and D: TNF-α inhibited club cell differentiation in vitro. E and F: IFN-γ reduced the number of basal cells and strongly diminished the number of ciliated and club cells. G–I: quantification of images (A–F) showing the frequency of Trp63+, Tubb4b+, and Pb1fa1+ cells under each condition. *P < 0.05, **P < 0.01. Values are means ± SD from 3 independent experiments. J–L: control and TNF-α- and IFN-γ-treated tracheal epithelial cells expressed Sox2 at the end of the 14-day treatment period. However, expression of the Sox2 marker appeared slightly reduced after treatment with recombinant IFN-γ. Images are representative of 3 independent experiments.