Figure 3.

Cep152 is required for centriole duplication. (A) U2OS cells were transfected with siRNAs against Cdk2 (positive control), Cep152 (O1 and O2), GL2 (negative control), and arrested in S phase by aphidicolin treatment. 70 h later, cells with more than four centrioles were counted. Red, Cep152; green, centrin-2; blue, DNA. (B) U2OS cells were transfected with either GL2 or Cep152 siRNAs (O1 or O2). Spindle poles were depicted with centrin staining (green, centrin-2) and mitotic spindles with α-tubulin antibodies (red) or DNA (blue). 72 h after transfection, cells with monopolar or bipolar mitotic spindles were counted. (C) U2OS cells were transfected with either GFP or GFP-Cep152 1–512 (green). Centrioles were visualized with centrin-2 staining (red). 48 h and 72 h after transfection, cells with less than two centrioles were counted. (left) Representative pictures of the observed phenotypes. (D) Cep152 full length (FL), Cep152 fragments (1–512 and 508–end), or GFP were overexpressed in U2OS cells. 48 h after transfection, cells with or without centriolar Plk4 staining (blue) were counted. GT335 (red; Bobinnec et al., 1998), an antibody to modified tubulin, was used as a marker for centrosomes. Insets show enlargements of centrosomes as merged image and individual channels. Error bars show the SDs of at least three independent experiments. Bars, 5 µm.