Figure 5.

Cep164 is involved in recruiting the distal appendage proteins NPHP1 and IFT88 to the M-centriole. (A) RPE1 cells stably expressing LAP-CSAP were treated with control or Cep164 siRNA for 24 h and serum starved for 48 h. Cells were stained for NPHP1 and Cep164. LAP-CSAP localizes to both centrioles and the axoneme and served as a centriolar and ciliary marker (Backer et al., 2012). (B) RPE1 cells, treated as in A, were stained for IFT88, Cep164, and polyglutamylated tubulin. In control depleted cells, IFT88 decorates the M-centriole of nonciliated cells and additionally the axoneme of ciliated cells. (C–E) Box-and-whisker plots showing NPHP1 (C), IFT88 (D), and Cep164 (E) signal intensity at the centrosome in RPE1 cells treated with the indicated siRNAs for 48 h and serum starved for another 24 h. (F) Western blot analysis of the experiment shown in C–E. Please note that the top and bottom panels are identical to Fig. 4 B, as these experiments were performed together. (G and H) Box-and-whisker plots show the relative signal intensity of NPHP1 (G) and IFT88 (H) at centrosomes in control and Cep164-depleted RPE1 cells expressing GFP or FLAG-Cep164-R1. Cells were treated as in Fig. 4 C. Knockdown efficiency was confirmed by Western blot analysis (Fig. 4 E). The left panels of A and B show merged images. Regions within the white boxes are shown at higher magnification to the right. A.U., arbitrary units.