FIGURE 3:

Ccdc11 is required for motile cilia formation in human multiciliated epithelial cells. (A) Schematic of the differentiation steps of cultured multiciliated respiratory epithelia. The key phases of the culture are depicted in cartoon form and include the progenitor phase (pre-ALI), centriologenesis (ALI day 7), initiation of ciliogenesis (ALI day 14), and mature multiciliated cells (ALI day 21). (B) Expression levels of endogenous Ccdc11 in hTECs. Cells were grown on Transwell permeable filter supports, and ALI was established 2 d after cells reached confluence. Cells were harvested on the indicated days, and lysates were analyzed by immunoblotting with antibodies against Ccdc11 and actin. Graph shows quantification of Ccdc11 levels normalized to actin for each stage. (C, D) Localization of endogenous Ccdc11 in hTEC at different stages of differentiation, viewed en face (C) or longitudinally (D). Progenitor cells were grown until confluent, and differentiation was induced by creating ALI. Samples were fixed on the indicated days and stained with antibodies against Ccdc11, centrin, acetylated tubulin, and ZO1 (to mark cell boundaries). Scale bar, 5 μm. (E) Colocalization of GFP-Ccdc11 and PCM-1 in hTEC. Progenitor cells were infected with lentivirus expressing GFP-Ccdc11 and grown until confluent, and differentiation was induced by creating ALI. Samples were fixed on the indicated days and stained with antibodies against GFP, centrin, and PCM-1. Scale bar, 5 μm. (F–H) Quantification of ciliogenesis defects upon loss of Ccdc11 in hTEC. shRNA-mediated depletion of Ccdc11 results in significant reduction of primary cilium assembly in pre-ALI progenitor cells (F), reduced number of multiciliated cells at ALI day 21 (G), and decreased number of cells expressing FoxJ1 at ALI day 21 (H). We scored 300 cells for each sample from three independent experiments; **p < 0.05.