Fig. 6

CCP5 and CCP6 are required to localize CP110 at mother centrioles in cycling cells. A Endogenous CP110 expression levels determined by immunoblotting in control (LacZ), CCP5, or CCP6 overexpressed HEK293T cells before and after serum starvation. In LacZ overexpressed cells, CP110 expression level was reduced after 24 h serum starvation, while in cells overexpressing CCP5 or CCP6, CP110 levels remain comparable before and after serum starvation. β-actin was used as a loading control. B In HEK293 cells treated with CCP5 or CCP6 siRNAs, the endogenous CP110 and CEP97 expression was reduced compared with that in control siRNA (siNC) treated cells. C Quantification of CP110 or CEP97 protein levels from results exemplified in B (3 independent experiments, Additional file 2). The intensities of the immunoblotting bands were normalized to those of β-actin. D hTERT-RPE1 cells transfected with control (siNC), CCP5, or CCP6 siRNA in the presence of serum were immunostained with γ-tubulin (green), ARL13B (red), and nuclei visualized with DAPI (blue). Depletion of CCP5 or CCP6 induced ciliation in cycling cells. E The elongated structure formed in CCP5 or CCP6 depleted hTERT-RPE1 cells in the presence of serum was positive for another cilia marker GT335 (green), but devoid of the centriole protein Centrin-1 (red). Nuclei were visualized with DAPI (blue). F Quantification of the ciliated cells in CCP5 or CCP6 depleted hTERT-RPE1 cells in the presence of serum as exemplified in D. Data were obtained from 3 independent experiments with at least 30 cells analyzed in each experiment per experimental condition (Additional file 2). G hTERT-RPE1 cells transfected with control (siNC), CCP5, or CCP6 siRNA in the presence of serum were immunostained with γ-tubulin (green), CP110 (red), and nuclei visualized with DAPI (blue). In control group, cells with 2 dots of CP110 were commonly seen, but in CCP5- or CCP6-depleted cells, those with only 1 dot of CP110 became more common, where γ-tubulin is still present as 2 dots, indicative of the integrity of centrioles. H Quantification of the percentage of cells with the indicated number of CP110 dots in hTERT-RPE1 cells transfected with control (siNC), CCP5, or CCP6 siRNA in the presence of serum (3 independent experiments; at least 25 cells analyzed per experimental condition in each experiment, Additional file 2). Error bars represent s.d., ∗ , P < 0.05; ∗  ∗ , P < 0.01 Student’s t test. Scale bars: 10 µm (D, E); 4 µm (H)