Fig. 1.

Centriole elongation is impaired in the absence of CEP295. (A) U2OS cells were transfected with control or CEP295-targeting siRNAs for 4 days. On day 3, aphidicolin was added to arrest cells at early S phase. After 24 h, the cells were released in fresh medium for another 8 h to enrich for G2-phase cells. The treated cells were fixed and stained with antibodies against CEP162 (green) and acetylated tubulin (Ac-tub, red). DNA was counterstained with DAPI. (B) Elongation of centrioles was calculated by measuring the distances between the centers of the two dots (representing staining for CEP162) within each pair of orthogonally arranged G2 centrioles. Error bars indicate the s.d. (standard deviation); ***P<0.0001 (two-tailed unpaired t-test). (C–G) PLK4-inducible cells were treated with sicontrol or siCEP295 as described in (C). IF, immunofluorescence; EM, electron microscopy; Dox, doxycycline. Cells were analyzed by immunofluorescence microscopy using the indicated antibodies (D), or by electron microscopy (E). Quantitative analysis of the centriole lengths (F) or the diameters (G) of daughter and mother centrioles acquired from electron microscopy images of sicontrol- and siCEP295-treated PLK4-inducible cells. Error bars represent the mean±s.d.; NS, not significant (two-tailed unpaired t-test). Arrows indicate reduced intensity of Ac-tub at the nascent centrioles.