Fig. 4

CCP5 and CCP6 differently regulate MT modifications in cilia. A qRT-PCR analysis confirmed that two independent siRNAs targeting human CCP5 (siCCP5#1, siCCP5#2) significantly reduced the CCP5 expression level in hTERT-RPE1 cells compared to cells transfected with non-targeting siRNA control (siNC). Data are means ± s.d. of 3 independent experiments (Additional file 2). B Immunoblotting analysis examined the efficiency of 2 CCP6 siRNAs (siCCP6#1, siCCP6#2) in depleting the overexpressed GFP-CCP6 in HEK293T cells. C Ciliary axoneme of hTERT-RPE1 cells treated with indicated siRNAs after 24-h serum starvation were detected with ARL13B (red) immunoreactivity for the length or GT335 or polyE (green) for the polyglutamylation levels. D Quantification of the ratio of ciliated cells showed that treatment with individual CCP5 or CCP6 siRNA did not affect cilia formation (3 independent experiments; at least 45 cells analyzed per experimental condition, Additional file 2). E–G Quantitative analysis of the length of cilia (E), axonemal GT335 (F), or polyE (G) in CCP5- or CCP6-depleted cells as exemplified in C. Each dot represents one cell. H Depletion of CCP5 or CCP6 differently increased axonemal glutamylation or polyglutamylation level as measured with GT335 (green) or polyE (red) immunoreactivity respectively. The length ratios between axonemal GT335 and polyE immunoreactivity were quantified in I (siNC: n = 45, siCCP5: n = 41, siCCP6: n = 31 cilia). Error bars represent s.d.. ∗  ∗ , P < 0.01, Student’s t test. Scale bars: 2 µm