FIGURE 4.

Regulation of TJs protein expression during hNECs differentiation. IL-13 decreased the mRNA expression of ZO-1, occludin, Cldn3, and Foxj1 but increased MUC5AC mRNA level in IL-13-matured hNECs as compared to untreated group (A–E). IF staining was performed for Cldn3, MUC5AC, and βIV-tubulin from Day 3 to 21 during hNECs differentiation (F,G). Cldn3 was only detected when ciliated cells were first observed at Day 11 and Cldn3 was co-stained with βIV-tubulin during hNECs differentiation. Cldn3 localization is weakly detectable at poorly ciliated areas of hNECs induced by IL-13 whereas Cldn3 localization was more widespread in regions with more fully ciliated cells in untreated hNECs. The relative target gene was normalized to 2^{– ΔCT} with RPL13A as a housekeeping gene. Two-way ANOVA was used to analyzed differences between hNECs with and without IL-13 treatment. Data were presented as median with an interquartile range. Scale bar = 20 μm. hNECs, n = 9.