Fig. 5. Ciliogenesis in the control and KD collagen I cysts. (A) Volocity 3D imaging showing ciliogenesis in different KD cysts. Ciliary staining (acetylated tubulin, green) from 1. Cav-1 KD, 2. Anx-13 KD, 3. Gal-3 KD, 4. Crb-3 KD, 5. Stx-3 KD and 6. VIP17 KD cysts. Scaling of the different cysts is shown as volumetric units (in µm) and is indicated separately for each of the image panels. (B) Primary cilia of the individual cells are visualized by staining for acetylated β-tubulin (green) and nuclei (blue, DAPI staining). Ciliary staining reveals two structural parts of these organelles, a punctated signal indicating the ciliary base at the apical surface of the individual cells and the outwards reaching ciliary processes projecting into the lumenal space. (C) Effect of different KDs on primary cilia formation and characteristic phenotypes of ciliogenesis in Anx-13, Cav-1, Crb-3, Gal-3, Stx-3 and VIP17 KD (1., 2., 3., 4., 5. and 6., respectively). Staining for actin (TRITC-phalloidin, red) and nuclei (DAPI, blue) show the overall structure and morphology of the KD cysts in comparison with the ciliary structures (AlexaFluor®488, green). Scale bars, 30 µm. (D) Percentage of cysts with normal (green bars) versus defective (red bars) ciliogenesis in the different KDs compared with the phenotype observed in control cysts. Examined were 100-200 cysts of each KD after 14 days of culture in collagen I gel with standard MEM supplemented with 5% FCS. Data are the mean ± s.d. (n=3). Statistical significance was analyzed by Student's t-test; ^**P<0.001, very significant; ^*P<0.01, significant.