FIGURE 4.

Knockdown of Cdc42 and Tuba inhibit ciliogenesis. A, MDCK cells containing stable shRNA-mediated knockdown of Cdc42 and Tuba were grown on Transwell filters for 14 days. Using an antibody against acetylated α-tubulin (red), ciliogenesis was examined by confocal microscopy combined with three-dimensional (3D) reconstruction of the stacked series. Ciliogenesis was virtually completely inhibited when Cdc42 was knocked down and significantly inhibited following Tuba knockdown. Again, please note that DAPI staining (blue) is at a different level in the cell than staining for acetylated α-tubulin but is included in the merged figure to delineate individual cells and allow for statistical analysis. Scale bar = 5 μm. B, quantification of ciliogenesis was performed using a ratio of cilia to cell nuclei. Significantly fewer cilia were seen in the MDCK cells following Cdc42 and Tuba knockdown. C, the same Cdc42 and Tuba knockdown MDCK cells were grown on Transwell filters for 14 days, fixed in glutaraldehyde, and then SE microscopy was performed (Phillips XL20). Confirming the results in (A), primary cilia were almost never seen following Cdc42 knockdown and were rarely seen following Tuba knockdown. Scale bar = 1.0 μm. D, quantification of ciliogenesis was again performed by counting the number of cilia per surface area, as individual cells could not be identified. Significantly fewer cilia were seen in the MDCK cells following Cdc42 and Tuba knockdown..