Cep63 and Cep152 Cooperate to Ensure Centriole Duplication
Figure 4
Cep63 and Cep152 are required for centrosome reduplication in mammalian cells.
(A) U2OS cells stably expressing GFP or GFP-Cep63 W (resistant to siRNA Cep63-2) were treated with Cep63 siRNAs 1 or 2 and incubated with 1.9 µg/ml aphidicolin (Aph) for 72 hours. GFP cells were stained with anti-Cep63 (green) and γ-tubulin (red). GFP-Cep63 W cells were stained with anti-γ-tubulin (red) and GFP-Cep63 W was detected by direct fluorescence. Scale bar 1 µm. (B) U2OS GFP-Cep63 W cell lysates were analysed by Western blotting with anti-GFP to detect GFP-Cep63 W and α-tubulin antibodies, used as a loading control. (C) Cells with greater than 2 γ-tubulin foci were recorded in 3 independent experiments, n = 150. T-test p values are indicated for comparison of control and siRNA Cep63-2 in both cell lines. (D-E) Cep63 homozygous gene-trap MEFs (Cep63T/T) show reduced centrosome reduplication induced by aphidicolin or Cdk1 inhibitor. (D) Cep63+/+ or Cep63T/T 3T3 immortalised MEFs incubated with aphidicolin (2 µg/ml) for 72 hours, stained with anti- γ-tubulin antibodies (green), centrin 3 antibodies (red) and DAPI (blue). Scale bar 5 µm. (H) Quantification of Cep63+/+ and Cep63T/T MEFs with greater than 2 γ-tubulin foci in untreated, Cdk1 inhibitor (RO-3306, 10 µM), or aphidicolin treated populations, n>150.