Fig. 8

Segregation of the dual function of CCP5 and CCP6 in ciliogenesis suppression and ciliary length control using Tet-On inducible expression system. A Experimental scheme for inducible overexpression of CCP5 or CCP6 before or after ciliogenesis. hTERT-RPE1 cells were cultured for 24 h and infected with Tet-on inducible CCP5 or CCP6 expression virus for 48 h. The cells were then changed to serum-free medium and continuously cultured for another 48 h. To induce CCP expression before ciliogenesis, doxycycline (Dox) was added at the beginning of infection (Before SS, red) and remained after serum starvation, while to induce CCP expression after ciliogenesis doxycycline was added upon serum starvation (After SS, blue). B hTERT-RPE1 cells were infected with virus-containing Tet-On controlled CCP5-GFP or GFP-CCP6 expressing plasmid and the expression was induced at the time points shown in A. Cells were immunostained with GFP (green) and ARL13B (red), and nuclei were visualized with DAPI (blue). C Quantification of the ciliation in GFP-positive cells representatively shown in B with the untreated hTERT-RPE1 cells used as the control. When CCP5 or CCP6 expression is induced before serum starvation, the ratios of ciliated cells were significantly reduced. When their expression was induced after serum starvation, the ratios of ciliated cells were not affected (3 independent experiments; at least 30 cells analyzed per experimental condition, Additional file 2). D Quantitative analysis of the cilia length of GFP-positive cells when CCP5 or CCP6 expression were induced after serum starvation (after SS) as shown in B. Each dot represents one cell (Additional file 2). Error bars represent s.d.. ∗  ∗ , P < 0.01. Student’s t test. Scale bars: 10 µm